Method of producing polyunsaturated fatty acids, novel biosynthesis genes, and novel plant expression constructs

ABSTRACT

The present invention relates to a method for the production of unsaturated fatty acids with at least two double bonds and/or a method for the production of triglycerides with an increased content of polyunsaturated fatty acids with at least two double bonds. The invention furthermore relates to the advantageous use of the nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 18 in the method and for generating a transgenic organism, preferably a transgenic plant or a transgenic microorganism, with an increased content of fatty acids, oils or lipids with unsaturated C 18 -, C 20 -, or C 22 -fatty acids.  
     The invention furthermore relates to novel desaturases with the [lacuna] in the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 11 or its homologs, derivatives and analogs, and to gene constructs encompassing these genes or their homologs, derivatives or analogs, and to their use alone or in combination with biosynthesis genes of polyunsaturated fatty acids as shown advantageously in SEQ ID NO: 7 and SEQ ID NO: 9.  
     In addition, the invention relates to isolated nucleic acid sequences; expression cassettes comprising the nucleic acid sequences, vectors and transgenic organisms comprising at least one nucleic acid sequence or one expression cassette. In addition, the invention relates to unsaturated fatty acids with at least two double bonds and to triglycerides with an increased content of unsaturated fatty acids with at least two double bonds, and to their use.  
     Moreover, the invention relates to multiexpression cassettes for seed-specific expression, and to vectors or organisms which encompass a desaturase gene alone or in combination with further desaturases with the sequence SEQ ID NO:7 and/or elongase genes with the sequence SEQ ID NO: 9 or its homologs, derivatives or analogs using said expression cassettes.

[0001] The present invention relates to a method for the production ofunsaturated fatty acids with at least two double bonds and/or a methodfor the production of triglycerides with an increased content ofpolyunsaturated fatty acids with at least two double bonds. Theinvention furthermore relates to the advantageous use of the nucleicacid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or 11 in themethod and for generating a transgenic organism, preferably a transgenicplant or a transgenic microorganism, with an increased content of fattyacids, oils or lipids with unsaturated C₁₈-, C₂₀-, or C₂₂-fatty acids.

[0002] The invention furthermore relates to novel desaturases with the[lacuna] in the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 andSEQ ID NO: 11 or its homologs, derivatives and analogs, and to geneconstructs encompassing these genes or their homologs, derivatives oranalogs, and to their use alone or in combination with biosynthesisgenes of polyunsaturated fatty acids as shown advantageously in SEQ IDNO: 7 and SEQ ID NO: 9.

[0003] In addition, the invention relates to isolated nucleic acidsequences; expression cassettes comprising the nucleic acid sequences,vectors and transgenic organisms comprising at least one nucleic acidsequence or one expression cassette. In addition, the invention relatesto unsaturated fatty acids with at least two double bonds and totriglycerides with an increased content of unsaturated fatty acids withat least two double bonds, and to their use.

[0004] Moreover, the invention relates to multiexpression cassettes forseed-specific expression, and to vectors or organisms which encompass adesaturase gene alone or in combination with further desaturases withthe sequence SEQ ID NO:7 and/or elongase genes with the sequence SEQ IDNO: 9 or its homologs, derivatives or analogs using said expressioncassettes.

[0005] A series of products and by-products of naturally occurringmetabolic processes in microorganisms, animal cells and plant cells hasutility for a wide array of industries, including the feed, food,cosmetics and pharmaceutical industries. These molecules, which arecollectively termed “fine chemicals”, also include, for example, lipidsand fatty acids, one representative class of which are thepolyunsaturated fatty acids. Fatty acids and triglycerides have amultiplicity of uses in the food industry, in animal nutrition, incosmetics and in the pharmacological sector. Depending on whether theytake the form of free saturated or unsaturated fatty acids or elsetriglycerides with an increased content of saturated or unsaturatedfatty acids, they are suitable for a variety of uses; thus, for example,polyunsaturated fatty acids (PUFAs) are added to baby formula forincreasing the nutritional value. Moreover, PUFAs have a positive effecton the cholesterol level in the blood of humans and are therefore usefulfor protection against heart disease. Thus, they are used in a varietyof dietetic foods or in medicaments.

[0006] Microorganisms which are particularly useful for the productionof PUFAs are microorganisms such as Thraustochytria or Schizochytriastrains, algae such as Phaeodactylum tricornutum or Crypthecodiniumspecies, ciliates such as Stylonychia or Colpidium, fungi such asMortierella, Entomophthora or Mucor. Through strain selection, a numberof mutant strains of the respective microorganisms have been developedwhich produce an array of desirable compounds including PUFAs. However,the selection of strains in which the production of a particularmolecule is improved is a time-consuming and difficult process.

[0007] Alternatively, fine chemicals can conveniently be produced viaproducing, on a large scale, plants which have been developed in such away that they produce the abovementioned PUFAs. Particularly well suitedplants for this purpose are oil crop plants which comprise large amountsof lipid compounds, such as oilseed rape, canola, linseed, soybean,sunflower, borage and evening primrose. However, other useful plantscomprising oils or lipids and fatty acids are also well suited asmentioned in the detailed description of this invention. By means ofconventional breeding, an array of mutant plants has been developedwhich produce a spectrum of desirable lipids and fatty acids, cofactorsand enzymes. However, the selection of novel plant cultivars in whichthe production of a particular molecule is improved is a time-consumingand difficult process or indeed impossible if the compound does notoccur naturally in the respective plant, as is the case ofpolyunsaturated C₁₈-, C₂₀-fatty acids and C₂₂-fatty acids and those withlonger carbon chains.

[0008] Owing to the positive properties of unsaturated fatty acids,there has been no lack of attempts in the past to make available geneswhich are involved in the synthesis of fatty acids or triglycerides forthe production, in various organisms, of oils with a modified content ofunsaturated fatty acids. Thus, WO 91/13972 and its U.S. equivalentdescribe a Δ9-desaturase. A Δ15-desaturase is claimed in WO 93/11245 anda Δ12-desaturase is claimed in WO 94/11516. Δ6-desaturases are describedin WO 93/06712, U.S. Pat. No. 5,614,393, WO 96/21022 and WO 99/27111.Further desaturases are described, for example, in EP-A-0 550 162, WO94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukeyet al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347,1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. A Δ6-palmitoylACP desaturase is described and claimed in WO 96/13591. However, thebiochemical characterization of the various desaturases is incomplete asyet since the enzymes, being membrane-bound proteins, can only beisolated and characterized with great difficulty (McKeon et al., Methodsin Enzymol. 71, 1981: 12141-12147, Wang et al., Plant Physiol. Biochem.,26, 1988: 777-792).

[0009] In yeasts, both a shift in the fatty acid spectrum towardunsaturated fatty acids and an increase in productivity have been found(see Huang et al., Lipids 34, 1999: 649-659, Napier et al., Biochem. J.,Vol. 330, 1998: 611-614). However, the expression of the variousdesaturases in transgenic plants did not show the desired success. Whilea shift in the fatty acid spectrum toward unsaturated fatty acids wasdemonstrated, it emerged that the synthesis performance of thetransgenic plants dropped drastically, i.e. only smaller amounts of oilswere isolated compared with the original plants.

[0010] Neither yeasts nor plants naturally produce polyunsaturated C₂₀-and/or C₂₂-fatty acids with at least two double bonds in the fatty acidmolecule, such as arachidonic acid (ARA) and/or eicosapentaenoic acid(EPA) and/or docosahexaenoic acid (DHA).

[0011] This is why there still exists a great demand for novel geneswhich encode enzymes which are involved in the biosynthesis ofunsaturated fatty acids and which make possible their production on anindustrial scale. None of the prior-art biotechnological methods for theproduction of polyunsaturated fatty acids yields the abovementionedfatty acids in economically useful quantities.

[0012] It is an object of the present invention to provide furtherenzymes for the synthesis of polyunsaturated fatty acids and to usethese enzymes, with or without other enzymes, in a method for theproduction of polyunsaturated fatty acids.

[0013] We have found that this object is achieved by the novel methodfor the production of fatty acid esters with an increased content ofpolyunsaturated fatty acids with at least two double bonds, whichcomprises introducing, into a fatty-acid-ester-producing organism, atleast one nucleic acid sequence selected from the group consisting of

[0014] a) a nucleic acid sequence with the sequence shown in SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11,

[0015] b) nucleic acid sequences which, owing to the degeneracy of thegenetic code, are obtained by backtranslating the amino acid sequencesshown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12,

[0016] c) derivatives of the nucleic acid sequence shown in SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11, which encodepolypeptides with the amino acid sequences shown in SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6 or SEQ ID NO: 12 and have at least 50% homology atthe amino acid level, without essentially reducing the enzymatic actionof the polypeptides,

[0017] growing the organism, and isolating the fatty acid esters presentin the organism.

[0018] The nucleic acid sequences used in the method according to theinvention are isolated nucleic acid sequences which encode polypeptideswith Δ5-, Δ6- or Δ12-desaturase activity.

[0019] It is advantageous to produce fatty acid esters withpolyunsaturated C₁₈-, C₂₀- and/or C₂₂-fatty acid molecules with at leasttwo double bonds in the fatty acid ester by the method according to theinvention. These fatty acid molecules preferably comprise three, four orfive double bonds and advantageously lead to the synthesis ofarachidonic acid (ARA), eicosapentaenoic acid (EPA) or docosahexaenoicacid (DHA).

[0020] The fatty acid esters with polyunsaturated C₁₈-, C₂₀- and/orC₂₂-fatty acid molecules can be isolated from the organisms used for theproduction of the fatty acid esters in the form of an oil or lipid forexample in the form of compounds such as sphingolipids,phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerides, diacyl glycerides, triacyl glycerides or other fatty acidesters comprising the polyunsaturated fatty acids with at least twodouble bonds.

[0021] Suitable organisms for the production by the method according tothe invention are, in principle, all prokaryotic or eukaryotic organismssuch as prokaryotic or eukaryotic microorganisms such as Gram-positiveor Gram-negative bacteria, fungi, yeasts, algae, ciliates, animal orplant cells, animals or plants such as mosses, dicotyledonous ormonocotyledonous plants. It is advantageous to use, in the methodaccording to the invention, organisms which belong to the oil-producingorganisms, that is to say which are used for the production of oils,such as microorganisms such as Crypthecodinium, Thraustochytrium,Phaeodactylum and Mortierella, Entomophthora, Mucor, and other algae orfungi, and animals or plants, in particular plants, preferably oil cropplants which contain large amounts of lipid compounds, such as soybean,peanut, oilseed rape, canola, sunflower, safflower, evening primrose,linseed, borage, trees (oil palm, coconut) or crops such as maize,wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper,Tagetes, solanaceous plants such as potato, tobacco, aubergine andtomato, Vicia species, pea, alfalfa or bush plants (coffee, cacao, tea),Salix species, and also perennial grasses and fodder crops. Plantsaccording to the invention which are especially preferred are oil cropplants such as soybean, peanut, oilseed rape, canola, linseed, eveningprimrose, sunflower, safflower or trees (oil palm, coconut).

[0022] The method according to the invention comprises either breeding asuitable transgenic organism or transgenic microorganism or breedingtransgenic plant cells, tissues, organs or intact plants comprising thenucleotide sequences according to the invention of SEQ ID NO: 1, 3, 5 or11, if appropriate in connection with the sequences shown in SEQ ID NO:7 and/or SEQ ID NO: 9 alone or in combination with sequences ofexpression constructs from SEQ ID NO: 13-17 or their homologs,derivatives or analogs or a gene construct which encompasses SEQ ID NO:1, 3, 5 or 11, if appropriate in connection with SEQ ID NO: 7 and/or 9or their homologs, derivatives or analogs, or a vector comprising thissequence or the gene construct which brings about the expression ofnucleic acid molecules according to the invention, so that a finechemical is produced. In a preferred embodiment, the process furthermorecomprises the step of obtaining a cell comprising such nucleic acidsequences according to the invention, wherein a cell is transformed witha desaturase nucleic acid sequence, a gene construct or a vector whichbring about the expression of a desaturase nucleic acid according to theinvention, alone or in combination. In a further preferred embodiment,this method furthermore comprises the step of obtaining the finechemical from the culture. In an especially preferred embodiment, thecell belongs to the order of the ciliates, to microorganisms such asfungi, or to the plant kingdom, in particular to oil crop plants;especially preferred are microorganisms or oil crop plants, for examplepeanut, oilseed rape, canola, linseed, soybean, safflower (thistle),sunflowers or borage.

[0023] Transgenic/recombinant is to be understood as meaning, for thepurposes of the invention, that the nucleic acids according to theinvention are not at their natural location in the genome of anorganism; it is possible here for the nucleic acids to be expressedhomologously or heterologously. However, transgenic/recombinant alsomeans that the nucleic acids according to the invention are at theirnatural location in the genome of an organism, but that the sequence hasbeen modified over the natural sequence and/or that the regulatorysequences of the natural sequences have been modified.Transgenic/recombinant preferably describes the expression of thenucleic acids according to the invention at an unnatural location in thegenome, that is to say a homologous or, preferably, heterologousexpression of the nucleic acids exists. Preferred transgenic organismsare the abovementioned transgenic plants, preferably oil crop plants.

[0024] The polyunsaturated fatty acids contained in the fatty acidesters produced by the method according to the invention can beliberated, for example, via treatment with alkali such as aqueous KOH orNaOH, advantageously in the presence of an alcohol such as methanol orethanol, and can be isolated via, for example, phase separation andsubsequent acidification, with, for example, H₂SO₄.

[0025] A further subject matter of the invention are oils, lipids and/orfatty acids containing at least two double bonds in the fatty acidmolecules, preferably three, four, five or six double bonds, which havebeen produced by the above-described method according to the invention.A further subject matter of the invention are also compositionscomprising the abovementioned oils, lipids and/or fatty acids, and theuse of the oils, lipids and/or fatty acids or of the compositions infeed, foodstuffs, cosmetics or pharmaceuticals.

[0026] A further aspect of the invention relates to methods ofmodulating the production of a molecule by a microorganism. Thesemethods encompass the contacting of the cell with a substance whichmodulates the desaturase activity according to the invention alone or incombination or the desaturase nucleic acid expression, so that acell-associated activity is modified in relation to the same activity inthe absence of the substance. In a preferred embodiment, one or twometabolic pathway(s) of the cell for lipids and fatty acids, cofactorsand enzymes is/are modulated, or the transport of compounds throughthese membranes is modulated, so that the yield or the production rateof a desired fine chemical by this microorganism is improved. Thesubstance which modulates the desaturase activity can be a substancewhich stimulates the desaturase activity or desaturase nucleic acidexpression or which can be used as intermediate in fatty acidbiosynthesis. Examples of substances which stimulate the desaturaseactivity or desaturase nucleic acid expression are, inter alia, smallmolecules, active desaturases and desaturase-encoding nucleic acidswhich have been introduced into the cell. Examples of substances whichinhibit the desaturase activity or desaturase expression are, interalia, small molecules and antisense desaturase nucleic acid molecules.

[0027] A further aspect of the invention relates to methods ofmodulating the yields of a desired compound from a cell, comprisingintroducing, into a cell, a wild-type or mutant desaturase gene which iseither maintained on a separate plasmid or integrated into the genome ofthe host cell. Upon integration into the genome, integration can berandom or can be effected by recombination in such a way that the nativegene is replaced by the copy introduced, thereby modulating theproduction of the desired compound by the cell, or by using a gene intrans, so that the gene is linked operably to a functional expressionunit comprising at least one sequence which ensures the expression of agene and at least one sequence which ensures the polyadenylation of afunctionally transcribed gene.

[0028] In a preferred embodiment, the yields are modified. In a furtherpreferred embodiment, the desired chemical is augmented, it beingpossible to reduce undesired interfering compounds. In an especiallypreferred embodiment, the desired fine chemical is a lipid or a fattyacid, a cofactor or an enzyme. In an especially preferred embodiment,this chemical is a polyunsaturated fatty acid. More preferably, it isselected from among arachidonic acid (ARA), eicosapentaenoic acid (EPA)or docosahexaenoic aicd (DHA).

[0029] The present invention provides novel nucleic acid molecules whichare suitable for identifying and isolating desaturases of PUFAbiosynthesis and which can be used for the modification of oils, fattyacids, lipids, lipid-derived compounds and, most preferably, for theproduction of polyunsaturated fatty acids.

[0030] The invention furthermore provides multiexpression cassettes andconstructs for the multiparallel seed-specific expression of genecombinations in plants. Microorganisms such as Crypthecodinium,Thraustochytrium, Phaeodactylum and Mortierella, Entomophthora andMucor, and other algae and fungi and plants, in particular oil cropplants, are preferred organisms for the method according to theinvention.

[0031] Cloning vectors and techniques for the genetic manipulation ofthe abovementioned microorganisms and ciliates, algae or relatedorgansisms such as Phaeodactylum tricornutum, are described in WO98/01572 or in Falciatore et al., 1999, Marine Biotechnology1(3):239-251, and Dunahay et al., 1995, Genetic transformation ofdiatoms, J. Phycol. 31:1004-1012 and the references cited therein.Thereby, the abovementioned nucleic acid molecules can be used in themethod according to the invention by modifying the organisms by means ofgenetic engineering so that they become better or more efficientproducers of one or more fine chemicals. This improved production orproduction efficiency of a fine chemical can be brought about by adirect effect of the manipulation of a gene according to the inventionor by an indirect effect of this manipulation. Fine chemicals for thepurposes of the invention are understood as being fatty acid esterscontaining polyunsaturated fatty acids with at least two double bonds,such as sphingolipids, phosphoglycerides, lipids, glycolipids,phospholipids, monoacyl glycerides, diacyl glycerides, triacylglycerides or other fatty acid esters containing the polyunsaturatedfatty acids with at least two double bonds. They are furthermoreunderstood as being compounds such as vitamins, for example vitamin E,vitamin C, vitamin B2, vitamin B6, pantolactone, carotenoids such asastaxanthin, β-carotene, zeaxanthin and others.

[0032] Mosses and algae are the only plant systems known which producesubstantial amounts of polyunsaturated fatty acids such as arachidonicacid (ARA) and/or eicosapentaenoic aicd (EPA) and/or docosahexaenoicacid (DHA). Mosses contain PUFAs in membrane lipids, while algae,organisms related to algae and some fungi also accumulate substantialamounts of PUFAs in the triacylglycerol fraction. Nucleic acid moleculeswhich are isolated from such strains which also accumulate PUFAs in thetriacylglycerol fraction are therefore particularly advantageouslysuitable for modifying the lipid and PUFA production system in a host,in particular in microorganisms, such as in the microorganisms mentionedabove, and plants such as oil crop plants, for example oilseed rape,canola, linseed, soybean, sunflowers, borage. They can therefore be usedadvantageously in the method according to the invention.

[0033] The nucleic acids according to the invention are thereforeparticularly advantageously suitable for isolating nucleic acids fromtriacylglycerol-accumulating microorganisms and for identifying such DNAsequences and the enzymes encoded by them in other species which aresuitable for modifying the biosynthesis of PUFA-precursor molecules inthe organisms in question.

[0034] Microorganisms such as Crypthecodinium cohnii, Thraustochytriumand Phaeodactylum species are microorganisms which are capable ofaccumulating PUFAs such as ARA, EPA or DHA in triacylglycerols.Thraustochytria are phylogenetically also closely related to strains ofSchizochytria. The ability to identify desaturases with reference to thenucleic acids according to the invention, for example predicting thesubstrate specificity of enzymes, can therefore be of enormoussignificance. Furthermore, these nucleic acid molecules can act asreference sequences for mapping related genomes or for deriving PCRprimers.

[0035] The nucleic acid molecules according to the invention encodeproteins termed desaturases. These desaturases can exert, for example, afunction involved in the metabolism (for example in the biosynthesis orthe degradation) of compounds required for the synthesis of lipids orfatty acids, such as PUFAs, or can participate in the transmembranetransport of one or more lipid/fatty acid compounds, either into thecell or out of the cell.

[0036] The nucleic acid sequences according to the invention encodedesaturases which are suitable for the production of long-chainpolyunsaturated fatty acids, preferably having more than sixteen,eighteen or twenty carbon atoms in the carbon skeleton of the fatty acidand/or at least two double bonds in the carbon chain, a nucleic acidaccording to the invention encoding an enzyme capable of introducingdouble bonds at the Δ5 position, in another case at the Δ6 position andin a further case at the Δ12 position. Large amounts of PUFAs may beobtained in the triacylglycerol fraction with the aid of these nucleicacids. Furthermore, further desaturases have been isolated which, aloneor together with a Δ4 desaturase, can be utilized for a method for theproduction of polyunsaturated fatty acids. In the application, thesingular, i.e. a desaturase gene or protein, is also understood asmeaning the plural, i.e. the desaturase genes or proteins.

[0037] The production of a trienoic acid with C₁₈-carbon chain with theaid of desaturases has already been demonstratead. However, in thesemethods known from the literature, the production of γ-linolenic acidwas claimed. As yet, however, nobody has been able to demonstrate theproduction of very long-chain polyunsaturated fatty acids (with C₂₀carbon chain and longer and of trienoic acids and higher unsaturatedtypes) by modified organisms alone.

[0038] To produce the long-chain PUFAs according to the invention, thepolyunsaturated C₁₈-fatty acids must first be elongated by at least twocarbon atoms by the enzymatic activity of an elongase. Following anelongation cycle, this enzyme activity leads to C₂₀-fatty acids, andafter two, three and four elongation cycles, to C₂₂-, C₂₄- or C₂₆-fattyacids. The nucleic acid sequences disclosed in the present inventionwhich encode various desaturases can, in concert with elongases, lead tovery long-chain polyunsaturated fatty acids. The activity of thedesaturases according to the invention preferably leads to C₁₈-, C₂₀-and/or C₂₂-fatty acids with at least two double bonds in the fatty acidmolecule, preferably with three, four, five or six double bonds,especially preferably to C₁₈- and/or C₂₀-fatty acids with at least twodouble bonds in the fatty acid molecule, preferably with three, four orfive double bonds in the molecule. The elongation of the fatty-acid canbe effected by combining the desaturases according to the invention withan elongase activity, it being possible to use the elongase encoded bySEQ ID NO: 9 in an advantageous fashion. After the elongation by theenzyme(s) according to the invention has taken place, furtherdesaturation steps such as, for example, a desaturation at the Δ5position, may take place. The combination with other elongases such asthose which lead to an elongation from C₁₈- to C₂₀-chains or from C₂₀-to C₂₂₋₂₄-chains as disclosed in WO 00/12720 may also be used and/or adesaturase with activity for the Δ4 position can advantageously beemployed in order to obtain the highly desaturated fatty acids. Theproducts of the desaturase activities and the possible furtherdesaturation therefore lead to preferred PUFAs with a higher degree ofdesaturation, such as dihomo-γ-linolenic acid, docosadienoic acid,arachidonic acid, ω6-eicosatrienedihomo-γ-linolenic acid,eicosapentaenoic acid, ω3-eicosatrienoic acid, ω3-eicosatetraenoic acid,docosapentaenoic acid or docosahexaenoic acid. Substrates of the enzymeactivity according to the invention are, for example, taxoleic acid;6,9-octadecadienoic acid, linoleic acid, pinolenic acid, α- orγ-linolenic acid or stearidonic acid and arachidonic acid,eicosatetraenoic acid, docosopentaenoic acid, eicosapentaenoic acid.Preferred substrates are linoleic acid, γ-linolenic acid and/orα-linolenic acid and arachidonic acid, eicosatetraenoic acid,docosapentaenoic acid, eicosapentaenoic acid. Especially preferredproducts of the process according to the invention are arachidonic acid,docosapentaenoic acid, eicosapentaenoic acid. The C₁₈-fatty acids withat least two double bonds in the fatty acid can be elongated by theenzymatic activity according to the invention in the form of the freefatty acid or in the form of the esters, such as phospholipids,glycolipids, sphingolipids, phosphoglycerides, monoacyl glycerides,diacyl glycerides or triacyl glycerides.

[0039] Of particular importance for human nutrition is the conjugatedlinoleic acid “CLA”. CLA is understood as meaning in particular fattyacids such as C18:2^(9 cis, 11trans) or the isomerC18:2^(10trans, 12 cis), which, once taken up, can be desaturated orelongated in the body owing to human enzyme systems and can contributeto health-promoting effects. The desaturases according to the invention(Δ12-desaturase) also allow those conjugated fatty acids with at leasttwo double bonds in the molecule to be desaturated and thus allow suchhealth-promoting fatty acids to be made available for human nutrition.Further examples of conjugated fatty acids are α-parinaric acid, punicicacid, eleostearic acid and calendulic acid.

[0040] Using cloning vectors in plants and in the transformation ofplants like those which are published and cited in: Plant MolecularBiology and Biotechnology (CRC Press, Boca Raton, Fla.), Chapter 6/7,pp. 71-119 (1993); F. F. White, Vectors for Gene Transfer in HigherPlants; in: Transgenic Plants, Vol. 1, Engineering and Utilization,Eds.: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al.,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilization, Eds.: Kung and R. Wu, Academic Press (1993), 128-143;Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991),205-225)), the nucleic acids according to the invention can be used forthe recombinant modification of a broad spectrum of plants so that thisplant becomes a better or more efficient producer of one or morelipid-derived products, such as PUFAs. This improved production orproduction efficiency of a lipid-derived product, such as PUFAs, can bebrought about by a direct action of the manipulation or an indirectionaction of this manipulation.

[0041] A series of mechanisms exist by means of which the modificationof a desaturase protein according to the invention can have a directeffect on the yield, production and/or production efficiency of a finechemical from an oil crop plant or a microorganism, owing to a modifiedprotein. The number or activity of the desaturase protein or desaturasegene and of gene combinations of desaturases and elongases can beincreased, so that larger amounts of these compounds are produced denovo since the organisms lacked this activity and ability tobiosynthesize them prior to introduction of the gene in question. Thisalso applies analogously to the combination with further desaturases orelongases or further enzymes of the lipid metabolism. The use of variousdivergent sequences, i.e. sequences which differ at the DNA sequencelevel, may also be advantageous, or else the use of promoters for geneexpression which makes possible a different temporal gene expression,for example as a function of the degree of maturity of the seed oroil-storing tissue.

[0042] The introduction of a desaturase gene according to the invention,or several saturase genes, into an organism, alone or in combinationwith other genes in a cell can not only increase the biosynthesis fluxtoward the end product, but also increase, or generate de novo, thecorresponding triacylglycerol composition. Likewise, the number oractivity of other genes which participate in the import of nutrientsrequired for the biosynthesis of one or more fine chemicals (for examplefatty acids, polar and neutral lipids) can be increased, so that theconcentration of the precursors, cofactors or intermediates within thecells or within the storage compartment is increased, thus furtherincreasing the ability of the cells to produce PUFAs as describedhereinbelow. Fatty acids and lipids themselves are desirable as finechemicals; by optimizing the activity or increasing the number of one ormore desaturases which participate in the biosynthesis of thesecompounds, or by destroying the activity of one or more desaturaseswhich participate in the breakdown of these compounds, it can bepossible to increase the yield, production and/or production efficiencyof fatty acid and lipid molecules from plants or microorganisms.

[0043] The mutagenesis of the desaturase gene(s) according to theinvention may furthermore lead to a desaturase protein with modifiedactivities which have a direct or indirect effect on the production ofone or more desired fine chemicals. For example, the number or activityof the desaturase gene(s) according to the invention may be increased sothat the normal metabolic wastes or by-products of the cell (possiblyincreased in quantity due to the overproduction of the desired finechemical) are exported efficiently before they can damage othermolecules or processes within the cell (which would decrease theviability of the cell) or to interfere with the fine chemicalbiosynthetic pathways (which would decrease the yield, production orproduction efficiency of the desired fine chemical). Furthermore, therelatively large intracellular quantities of the desired fine chemicalmay themselves be toxic to the cell or interfere with enzyme feedbackmechanisms, such as allosteric regulation; for example, by increasingthe activity or number of other downstream enzymes or detoxificationenzymes of the PUFA pathway, it might increase the allocation of thePUFA to the triacylglycerol fraction; the viability of seed cells mightbe increased, in turn leading to better development of cultured cells orto seeds which produce the desired fine chemical. The desaturaseaccording to the invention may also be manipulated in such a way thatthe relative amounts of the various lipid and fatty acid molecules areproduced. This may have a profound effect on the lipid composition ofthe membrane of the cell and generates novel oils in addition to theoccurrence of newly-synthesized PUFAs. Since each type of lipid hasdifferent physical properties, a change in the lipid composition of amembrane may significantly alter membrane fluidity. Changes in membranefluidity can have an effect on the transport of molecules across themembrane and on the integrity of the cell, both of which have a profoundeffect on the production of fine chemicals. In plants, these changes mayadditionally impact on other traits, such as tolerance to abiotic andbiotic stress situations.

[0044] Biotic and abiotic stress tolerance is a general trait which itis desired to transmit to a broad spectrum of plants such as maize,wheat, rye, oats, triticale, rice, barley, soybeans, peanut, cotton,linseed, flax, oilseed rape and canola, cassava, pepper, sunflower andTagetes, solanaceous plants such as potato, tobacco, egg-plant andtomato, Vicia species, pea, alfalfa, bush plants (coffee, cacao, tea),Salix species, trees (oil palm, coconut) and perennial grasses andfodder crops. Being a further embodiment of the invention, these cropsare also preferred target plants for genetic engineering. Especiallypreferred plants according to the invention are oil crop plants such assoybean, peanut, oilseed rape, canola, sunflower, linseed, safflower,trees (oil palm, coconut) or crops such as maize, wheat, rye, oats,triticale, rice, barley, alfalfa, or bush plants (coffee, cacao, tea).

[0045] Accordingly, an aspect of the invention relates to isolatednucleic acid molecules (for example cDNAs) encompassing nucleotidesequences which encode one desaturase or several desaturases orbiologically active parts thereof, or nucleic acid fragments which aresuitable as primers or hybridization probes for detecting or amplifyingdesaturase-encoding nucleic acids (for example DNA or mRNA). Inespecially preferred embodiments, the nucleic acid molecule encompassesone of the nucleotide sequences shown in sequence ID NO:1 or 3 and 5 orthe coding region or a complement of one of these nucleotide sequences.In other especially preferred embodiments, the isolated nucleic acidmolecule according to the invention encompasses a nucleotide sequencewhich hybridizes with a nucleotide sequence as shown in the sequence SEQID NO: 1, 3, 5 or 11 or a portion thereof or which has at least 50%homology, preferably at least approxiately 60% homology, more preferablyat least approximately 70%, 80% or 90% homology and very especiallypreferably at least approximately 95%, 96%, 97%, 98%, 99% or morehomology therewith. In other preferred embodiments, the isolated nucleicacid molecule encodes one of the amino acid sequences shown in sequenceSEQ ID NO: 2, 4, 6 or 12. The preferred desaturase gene according to theinvention preferably also has at least one of the desaturase activitiesdescribed herein.

[0046] In a further embodiment, the isolated nucleic acid moleculeencodes a protein or a portion thereof, the protein or the portionthereof comprising an amino acid sequence which has sufficient homologywith an amino acid sequence of the sequence SEQ ID NO: 2, 4, 6 or 12that the protein or the portion thereof retains a desaturase activity.Preferably, the protein or the portion thereof encoded by the nucleicacid molecule retains the ability of participating in the metabolism ofcompounds required for the synthesis of cell membranes of plants or inthe transport of molecules across these membranes. In one embodiment,the protein encoded by the nucleic acid molecule has at leastapproximately 50% homology, preferably at least approximately 60%homology, more preferably at least approximately 70%, 80% or 90% andvery especially preferably at least approximately 95%, 96%, 97%, 98%,99% or more homology with an amino acid sequence of the sequence SEQ IDNO: 2, 4, 6 or 12. In a further preferred embodiment, the protein is afull-length protein which is essentially in parts homologous to acomplete amino acid sequence of SEQ ID NO: 2, 4, 6 or 12 (which isderived from the open reading frame shown in SEQ ID NO: 1, 3, 5 or 11).

[0047] In other embodiments, the isolated desaturase encompasses anamino acid sequence which has at least approximately 50% homology withone of the amino acid sequences of SEQ ID NO: 2, 4, 6 or 12 and whichcan participate in the metabolism of compounds required for thesynthesis of fatty acids in a microorganism or plant cell or in thetransport of molecules across these membranes, desaturated C₁₈- orC₂₀₋₂₂-carbon chains being understood with double bonds in at least twopositions.

[0048] In another preferred embodiment, the isolated nucleic acidmolecule originates from Phaeodactylum tricornutum UTEX646 and encodes aprotein (for example a desaturase fusion protein) containing abiologically active domain which has at least approximately 50% or morehomology with an amino acid sequence of the sequence SEQ ID NO: 2, 4, 6or 12 and retains the ability of participating in the metabolism ofcompounds required in the synthesis of cell membranes of plants or inthe transport of molecules across these membranes or has at least one ofthe desaturation activities resulting in PUFAs such as GLA, ALA,dihomo-γ-linolenic acid, ARA, EPA or DHA or their precursor molecules,and also encompasses heterologous nucleic acid sequences which encode aheterologous polypeptide or regulatory proteins.

[0049] As an alternative, the isolated desaturase can comprise an aminoacid sequence which is encoded by a nucleotide sequence which hybridizeswith a nucleotide sequence of SEQ ID NO: 1, 3, 5 or 11, for exampleunder stringent conditions, or which has at least approximately 50%homology, preferably at least approximately 60% homology, morepreferably at least approximately 70%, 80% or 90% homology and even morepreferably at least approximately 95%, 96%, 97%, 98%, 99% or morehomology therewith. It is also preferred for the preferred desaturaseforms likewise to have one of the desaturase activities describedherein.

[0050] In another embodiment, the isolated nucleic acid molecule is atleast 15, 25, 50, 100, 250 or more nucleotides in length and hybridizesunder stringent conditions with a nucleic acid molecule comprising anucleotide sequence of SEQ ID NO: 1, 3, 5 or 17. Preferably, theisolated nucleic acid molecule corresponds to a naturally occurringnucleic acid molecule. More preferably, the isolated nucleic acidmolecule encodes naturally occurring Phaeodactylum desaturase or abiologically active portion thereof.

[0051] A further embodiment of the invention are expression cassetteswhich make possible the expression of the nucleic acids according to theinvention with the sequences SEQ ID NO: 1, 3, 5 or 11 in. the variousorganisms such as microorganisms, for example bacteria, fungi, yeasts,ciliates, algae or animal or plant cells, or in animals or plants.

[0052] The expression cassette (=nucleic acid construct or fragment)according to the invention is to be understood as meaning the sequencesmentioned in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11which are the result of the genetic code and/or the functional ornonfunctional derivatives thereof which had been linked functionally toone or more regulatory signals for advantageously increasing geneexpression and which govern the expression of the coding sequence in thehost cell. These regulatory sequences are intended to make possible thetargeted expression of the genes and of protein expression. Depending onthe host organism, this may mean, for example, that the gene is firstinduced and only then expressed and/or overexpressed, or that it isexpressed and/or overexpressed immediately. For example, theseregulatory sequences are sequences to which inductors or repressorsbind, thus regulating the expression of the nucleic acid. In addition tothese novel regulatory sequences, or instead of these sequences, thenatural regulation of these sequences before the actual structural genesmay still be present and, if appropriate, may have been geneticallymodified so that the natural regulation has been eliminated and geneexpression increased. However, the gene construct may also have asimpler construction, that is to say no additional regulatory sequenceswere inserted before the nucleic acid sequence or its derivatives, andthe natural promoter together with its regulation has not been removed.Instead, the natural regulatory sequence was mutated in such a way thatregulation no longer takes place and/or gene expression is increased.These modified promoters can be inserted before the natural gene in theform of part-sequences (=promoter with parts of the nucleic acidsequences according to the invention) or else alone, in order toincrease the activity. Moreover, the gene construct can additionallyadvantageously also comprise one or more of what are known as enhancersequences linked functionally to the promoter, and these make possiblean increased expression of the nucleic acid sequence. Additionaladvantageous sequences, such as further regulatory elements orterminators, may also be inserted at the 3′ end of the DNA sequences.The Δ5 desaturase/Δ6 desaturase and/or Δ12 desaturase genes may bepresent in the expression cassette (=gene construct) in one or morecopies.

[0053] As described above, the regulatory sequences or factors canpreferably have a positive effect on the gene expression of theintroduced gene, thus increasing it. Thus, an enhancement of theregulatory elements can advantageously take place at transcriptionlevel, by using strong transcription signals such as promoters and/orenhancers. In addition, however, enhanced translation is also possible,for example by improving the stability of the mRNA.

[0054] A further aspect of the invention relates to vectors, for examplerecombinant expression vectors, comprising at least one nucleic acidmolecule according to the invention, and to host cells into which thesevectors have been introduced, in particular microorganisms, plant cells,plant tissues, plant organs or intact plants. In one embodiment, such ahost cell can store fine chemical compounds, in particular PUFAs; toisolate the desired compound, the cells are harvested. The compound(oils, lipids, triacyl glycerides, fatty acids) or the desaturase canthen be isolated from the medium or from the host cell which, in thecase of plants, are cells comprising or storing the fine chemicals, mostpreferably cells of storage tissues such as seed coats, tubers,epidermis cells and seed cells, endosperm or embryo tissue.

[0055] Yet another aspect of the invention relates to a geneticallymodified transgenic plant, preferably an oil crop plant as mentionedabove, especially preferably an oilseed rape or linseed plant into whicha desaturase gene has been introduced. In one embodiment, the genome ofoilseed rape or linseed has been modified by introducing, as transgene,a nucleic acid molecule according to the invention which encodes awild-type or mutated desaturase sequence. In another embodiment, anendogenous desaturase gene in the genome of the donor organismPhaeodactylum has been destroyed functionally by mutagenesis anddetection by means of DNA sequences or has been repressed by means ofantisense technology. In a preferred embodiment, oilseed rape or linseedis also used for the production of a desired compound such as lipids andfatty acids, with PUFas being especially preferred.

[0056] In yet another preferred embodiment, the moss Physcomitrellapatens can be used for demonstrating the function of a desaturase geneusing homologous recombination on the basis of the nucleic acidsdescribed in the present invention.

[0057] Yet another aspect of the invention relates to an isolateddesaturase gene or a part, for example a biologically active part,thereof. In a preferred embodiment, the isolated desaturase or a partthereof can participate in the metabolism of compounds required for thesynthesis of cell membranes in a microorganism or a plant cell or in thetransport of molecules via its membranes. In a further preferredembodiment, the isolated desaturase or the part thereof has sufficienthomology with an amino acid sequence of SEQ ID NO: 2, 4, 6 or 12 toretain the ability to participate in the metabolism of compoundsrequired for the synthesis of cell membranes in microorganisms or plantcells or in the transport of molecules via these membranes.

[0058] The invention also provides an isolated preparation of adesaturase in the form of a crude extract or as a pure protein.

[0059] The desaturase polypeptide or a biological active part thereofcan advantageously be linked functionally to a further polypeptide whichhas an enzymatic activity other than the desaturases, for example anelongase, acyltransferase or other activity, to form a fusion protein.This fusion protein advantageously has an activity which differs fromthat of the desaturase alone. In other preferred embodiments, thisfusion protein participates in the metabolism of compounds which arerequired for the synthesis of lipids and fatty acids, cofactors andenzymes in microorganisms or plants, or in the transport of moleculesvia these membranes. Especially preferably, the introduction of thisfusion protein into a host cell modulates the production of a desiredcompound within a cell and by the cell. In a preferred embodiment, thesefusion proteins also contain Δ4-, Δ5- or Δ6-, Δ8-, Δ15-, Δ17- orΔ19-desaturase activities, alone or in combination. Preferredembodiments are, in particular, those gene combinations which areselected from among SEQ ID NO: 7 or 9, or parts thereof, derivatives ortheir homologs. Particularly preferred are those combinations whichcontain the complete protein activity as in SEQ ID NO: 1, 3, 5 or 11and, inserted into multiexpression cassettes defined by SEQ ID NO: 13,14, 15, 16 and 17, are suitable for the transformation of plants andexpression in plants.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The invention relates to (an) isolated nucleic acid sequence(s)encoding a polypeptide with desaturase activity selected from the groupconsisting of

[0061] a) a nucleic acid sequence with the sequence shown in SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11,

[0062] b) nucleic acid sequences which, owing to the degeneracy of thegenetic code, are obtained by backtranslating the amino acid sequencesshown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12,

[0063] c) derivatives of the nucleic acid sequence shown in SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11, which encodepolypeptides with the amino acid sequences shown in SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6 or SEQ ID NO: 12 and have at least 50% homology atthe amino acid level, without essentially reducing the enzymatic actionof the polypeptides.

[0064] The invention furthermore relates to (an) amino acid sequence(s)which is/are encoded by the abovementioned nucleic acid sequence(s) (forthe purposes of the invention, the singular is intended to comprise theplural and vice versa). Specifically, the invention relates to aminoacid sequences encoded by the sequence shown in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5 or SEQ ID NO: 11.

[0065] The present invention provides nucleic acids and proteinmolecules with desaturase activity which participate in the metabolismof lipids and fatty acids, PUFA cofactors and enzymes in the mossPhyscomitrella patens or in the transport of lipophilic compounds viamembranes. The compounds according to the invention can be used formodulating the production of fine chemicals from organisms, for examplemicroorganisms, such as ciliates, fungi, yeasts, bacteria, algae and/orplants such as maize, wheat, rye, oats, triticale, rice, barley,soybean, peanut, cotton, Linum species such as linseed or flax, Brassicaspecies such as oilseed rape, canola and turnip rape, pepper, sunflower,borage, evening primrose and Tagetes, Solanaceae plants such as potato,tobacco, egg-plant and tomato, Vicia species, pea, cassava, alfalfa,bush plants (coffee, cacao, tea), Salix species, trees (oil palm,coconut) and perennial grasses and fodder crops, either directly (forexample when the overexpression or optimization of a fatty acidbiosynthesis protein has a direct effect on the yield, production and/orproduction efficiency of the fatty acid from modified organisms) or theycan have an indirect effect which nevertheless leads to an increasedyield, production and/or production efficiency of the desired compoundor to a decrease in undesired compounds (for example when the modulationof the metabolism of lipids and fatty acids, cofactors and enzymes leadsto changes in yield, production and/or production efficiency or thecomposition of the desired compound within the cells, which, in turn,may have an effect on the production of one or more fine chemicals).Aspects of the invention are illustrated in greater detail hereinbelow.

[0066] I. Fine Chemicals and PUFAs

[0067] The term “fine chemical” is known in the art and encompassesmolecules which have been produced by an organism and which are used ina variety of industries such as, by way of example but not by way oflimitation, the pharmaceuticals industry, agro industry, food industryand cosmetics industry. These compounds encompass lipids, fatty acids,cofactors and enzymes and the like (as described, for example, inKuninaka, A. (1996) Nucleotides and related compounds, pp. 561-612, inBiotechnology Vol. 6, Rehm et al., Ed., V C H Weinheim and referencescited therein), lipids, saturated and unsaturated fatty acids (forexample arachidonic acid), vitamins and cofactors (as described inUllmann's Encyclopedia of Industrial Chemistry, Vol. A27, Vitamins, pp.443-613 (1996) V C H Weinheim and references cited therein; and Ong, A.S., Niki, E., & Packer, L. (1995) Nutrition, Lipids, Health and DiseaseProceedings of the UNESCO/Confederation of Scientific and TechnologicalAssociations in Malaysia and the Society for Free Radical Research—Asia,held Sep. 1-3, 1994, in Penang, Malaysia, AOCS Press (1995)), enzymesand all other chemicals described by Gutcho (1983) in Chemicals byFermentation, Noyes Data Corporation, ISBN: 0818805086, and referencescited therein. The metabolism and the uses of certain fine chemicals areillustrated in greater detail hereinbelow.

[0068] The combination of various precursor molecules and biosyntheticenzymes leads to the production of various fatty acid molecules, whichhas a decisive effect on membrane composition. It can be assumed thatPUFAs are not only just incorporated into triacylglycerol, but also intomembrane lipids.

[0069] Membrane synthesis is a well characterized process in which anumber of components, including lipids as part of the bilayer membrane,are involved. The production of novel fatty acids such as PUFAs cantherefore generate novel properties of membrane functions within a cellor an organism.

[0070] Cell membranes serve a multiplicity of functions in a cell. Firstand foremost, a membrane delimits the contents of a cell from theenvironment, thus imparting integrity to the cell. Membranes can alsoact as barriers against the influx of dangerous or undesired compoundsor else against the efflux of desired compounds.

[0071] For more detailed descriptions and involvements of membranes andthe mechanisms involved, see Bamberg, E., et al. (1993) Charge transportof ion pumps on lipid bilayer membranes, Q. Rev. Biophys. 26:1-25;Gennis, R. B. (1989) Pores, Channels and Transporters, in: Biomembranes,Molecular Structure and Function, Springer: Heidelberg, pp. 270-322; andNikaido, H., and Saier, H. (1992) Transport proteins in bacteria: commonthemes in their design, Science 258:936-942, and the citations containedin each of these references.

[0072] Lipid synthesis can be divided into two parts: the synthesis offatty acids and their binding to sn-glycerol-3-phosphate, and theaddition or modification of a polar head group. Customary lipids used inmembranes encompass phospholipids, glycolipids, sphingolipids andphosphoglycerides. Fatty acid synthesis starts with the conversion ofacetyl-CoA either into malonyl-CoA by acetyl-CoA carboxylase or intoacetyl-ACP by acetyl transacylase. After a condensation reaction, thesetwo product molecules together form acetoacetyl-ACP, which is convertedvia a series of condensation, reduction and dehydration reactions togive a saturated fatty acid molecule with the desired chain length. Theproduction of the unsaturated fatty acids from these molecules iscatalyzed by specific desaturases, either aerobically by means ofmolecular oxygen or anaerobically (as regards fatty acid synthesis inmicroorganisms, see F. C. Neidhardt et al. (1996) E. coli andSalmonella. ASM Press: Washington, D.C., pp. 612-636 and referencescontained therein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes.Thieme: Stuttgart, New York, and the references contained therein, andMagnuson, K., et al. (1993) Microbiological Reviews 57:522-542 and thereferences contained therein).

[0073] Examples of precursors for PUFA biosynthesis are oleic acid,linoleic acid and linolenic acid. These C₁₈ carbon fatty acids must beelongated to C₂₀ and C₂₂ to give fatty acids of the eicosa and docosachain type. Various desaturases such as enzymes which haveΔ12-desaturase, Δ15-desaturase, Δ6-desaturase, Δ5- and Δ4-desaturaseactivity, can lead to arachidonic acid, eicosapentaenoic acid anddocosahexaenoic acid and various other long-chain PUFAs which can beextracted and used for various purposes in food and feed, cosmetic orpharmaceutical applications.

[0074] To produce long-chain PUFAs, the polyunsaturated C₁₈- orC₂₀-fatty acids must be polydesaturated as mentioned above. The nucleicacid sequences according to the invention encode first functionallyactive desaturases from Phaeodactylum tricornutum, a microorganismcomprising PUFAs in the triacylglycerol fraction. Double bonds can beintroduced into the Δ5, Δ6 or Δ12 position with the desaturasesaccording to the invention. The activities of the desaturases accordingto the invention preferably lead to C₁₈-+C₂₀-fatty acids with at leasttwo, three, four or five double bonds in the fatty acid molecule,preferably to C₂₀-fatty acids with, advantageously, three, four or fivedouble bonds in the fatty acid molecule. Desaturation can be effectedbefore or after elongation of the fatty acid in question. The productsof the desaturase activities and of the possible further desaturationand elongation therefore lead to preferred PUFAs with a higher degree ofdesaturation, including a further elongation of C₂₀- to C₂₂-fatty acids,to fatty acids such as linoleic acid, docosadienoic acid,dihomo-γ-linolenic acid, arachidonic acid,ω6-eicosatrienedihomo-γ-linolenic acid, eicosapentaenoic acid,ω3-eicosatrienoic acid, ω3-eicosatetraenoic acid, docosapentaenoic acidor docosahexaenoic acid. Preferred substrates of this enzyme activityaccording to the invention are taxoleic acid, 6,9-octadecadienoic acid,oleic acid, linoleic acid, γ-linolenic acid, pinolenic acid, α-linolenicacid, arachidonic acid, eicosapentaenoic acid or stearidonic acid.Preferred substrates are linoleic acid, γ-linolenic acid and/orα-linolenic acid, dihomo-γ-linolenic acid or arachidonic acid,eicosatetraenoic acid or eicosapentaenoic acid. The C₁₈- or C₂₀-fattyacids with at least two double bonds in the fatty acid can be elongatedby the enzyme activity according to the invention in the form of thefree acid or in the form of the esters, such as phospholipids,glycolipids, sphingolipids, phosphoglycerides, monoacyl glycerides,diacyl glycerides, triacyl glycerides or other esters.

[0075] Furthermore, fatty acids must subsequently be transported tovarious locations of modification and incorporated into thetriacylglycerol storage lipid. Another important step in lipid synthesisis the transfer of fatty acids to the polar head groups, for example byglycerol fatty acid acyl transferase (see Frentzen, 1998, Lipid,100(4-5):161-166).

[0076] For publications on plant fatty acid biosynthesis, desaturation,lipid metabolism and the membrane transport of fatty compounds,beta-oxidation, fatty acid modification and cofactors, triacylglycerolstorage and assembly including the references cited therein, see thefollowing articles: Kinney, 1997, Genetic Engeneering, Ed.: J K Setlow,19:149-166; Ohlrogge and Browse, 1995, Plant Cell 7:957-970; Shanklinand Cahoon, 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:611-641;Voelker, 1996, Genetic Engineering, Ed.: J K Setlow, 18:111-13;Gerhardt, 1992, Prog. Lipid R. 31:397-417; Gühnemann-Schäfer & Kindl,1995, Biochim. Biophys Acta 1256:181-186; Kunau et al., 1995, Prog.Lipid Res. 34:267-342; Stymne et al., 1993, in: Biochemistry andMolecular Biology of Membrane and Storage Lipids of Plants, Ed.: Murataand Somerville, Rockville, American Society of Plant Physiologists,150-158, Murphy & Ross 1998, Plant Journal. 13(1):1-16.

[0077] Vitamins, cofactors and nutraceuticals, such as PUFAs, encompassa group of molecules which higher animals can no longer synthesize andtherefore have to take up, or which higher animals can no longersynthesize themselves to a sufficient degree and must therefore take upadditionally, even though they are readily synthesized by otherorganisms such as bacteria. The biosynthesis of these molecules inorganisms which are capable of producing them, such as in bacteria, hasbeen largely characterized (Ullmann's Encyclopedia of IndustrialChemistry, “Vitamins”, Vol. A27, pp. 443-613, V C H Weinheim, 1996;Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry andMolecular Biology, John Wiley & Sons; Ong, A. S., Niki, E., & Packer, L.(1995) “Nutrition, Lipids, Health and Disease” Proceedings of theUNESCO/Confederation of Scientific and Technological Associations inMalaysia and the Society for Free Radical Research Asia, held Sep. 1-3,1994, in Penang, Malaysia, AOCS Press, Champaign, Ill. X, 374 pp.).

[0078] The abovementioned molecules are either biologically activemolecules themselves or precursors of biologically active substanceswhich act either as electron carriers or as intermediates in amultiplicity of metabolic pathways. Besides their nutritional value,these compounds also have a significant industrial value as colorants,antioxidants and catalysts or other processing auxiliaries. (For areview over structure, activity and industrial applications of thesecompounds, see, for example, Ullmann's Encyclopedia of IndustrialChemistry, “Vitamins”, Vol. A27, pp. 443-613, V C H Weinheim, 1996).Polyunsaturated fatty acids have a variety of functions andhealth-promoting effects, for example in the case of coronary heartdisease, inflammatory mechanisms, children's nutrition and the like. Forpublications and references including the references cited therein, see:Simopoulos, 1999, Am. J. Clin. Nutr. 70 (3rd Suppl.):560-569, Takahataet al., Biosc. Biotechnol. Biochem. 1998, 62(11):2079-2085, Willich andWinther, 1995, Deutsche Medizinische Wochenschrift 120(7):229 et seq.

[0079] II. Elements and Processes of the Invention

[0080] The present invention is based, inter alia, on the discovery ofnovel molecules termed herein desaturase nucleic acid and desaturaseprotein molecules, which exert an effect on the production of cellmembranes and lipids in Phaeodactylum tricornutum and, for example, havean effect on the movement of molecules via these membranes. In oneembodiment, the desaturase molecules participate in the metabolism ofcompounds required for the synthesis of cell membranes in organisms,such as microorganisms and plants, or indirectly affect the transport ofmolecules via these membranes. In a preferred embodiment, the activityof the desaturase molecules according to the invention for regulatingthe production of membrane components and membrane transport has aneffect on the production of the desired fine chemical by this organism.In an especially preferred embodiment, the activity of the desaturasemolecules according to the invention is modulated so that the yield,production and/or production efficiency of the metabolic pathways ofmicroorganisms or plants which regulate the desaturases acording to theinvention are modulated and the transport efficiency of compoundsthrough the membranes is modified, which either directly or indirectlymodulates the yield, production and/or production efficiency of adesired fine chemical by microorganisms and plants.

[0081] The term “desaturase” or “desaturase polypeptide” encompassesproteins which participate in the desaturation of fatty acids. Examplesof desaturases are disclosed in SEQ ID NO: 1, 3, 5, 11 or theirhomologues, derivatives or analogs. The terms desaturase or desaturasenucleic acid sequence(s) encompass nucleic acid sequences which encode adesaturase and part of which can be a coding region and alsocorresponding 5′- and 3′-untranslated sequence regions. Examples ofdesaturase genes are those shown in SEQ ID NO: 1, 3, 5 or 11. The termsproduction and productivity are known in the art and encompass theconcentration of the fermentation product (for example of the desiredfine chemical) which is formed within a specific period and in aspecific fermentation volume (for example kg product per hour perliter). The term production efficiency encompasses the time required forachieving a particular production quantity (for example the timerequired by the cell to establish its particular throughput rate of afine chemical). The term yield or product/carbon yield is known in theart and encompasses the efficiency with which the carbon source isconverted into the product (i.e. the fine chemical). This is usuallyexpressed as, for example, kg product per kg carbon source. Increasingthe yield of production of the compound increases the amount of themolecules obtained or of the suitable molecules of this compoundobtained in a specific quantity of culture over a defined period. Theterms biosynthesis or biosynthetic pathway are known in the art andencompass the synthesis of a compound, preferably of an organiccompound, by a cell from intermediates, for example in a multi-stepprocess which is subject to strong regulation. The terms catabolism orcatabolic pathway are known in the art and encompass the cleavage of acompound, preferably of an organic compound, by a cell into catabolytes(in general terms, smaller or less complex molecules), for example in amulti-step process which is subject to strong regulation. The termmetabolism is known in the art and encompasses the totality of thebiochemical reactions which take place in an organism. The metabolism ofa certain compound (for example the metabolism of a fatty acid) thusencompasses the totality of the biosynthetic, modification and catabolicpathways of this compound in the cell which are relevant to thiscompound.

[0082] In another embodiment, the nucleic acid sequences according tothe invention which encode desaturase molecules can modulate theproduction of a desired molecule, such as a fine chemical, in amicroorganism or in plants. There exist a series of mechanisms by whichthe modification of a sequence according to the invention can directlyaffect the yield, production and/or production efficiency of a finechemical from a microorganism strain or plant strain comprising thismodified protein. The number or activity of desaturases participating inthe transport of molecules of fine chemicals within, or out of, the cellcan be increased, so that greater amounts of these compounds aretransported via membranes, from which they can be obtained and convertedinto each other with greater ease. Furthermore, fatty acids,triacylglycerols and/or lipids are desirable fine chemicals themselves;optimizing the activity or increasing the number of one or moredesaturases according to the invention which participate in thebiosynthesis of these compounds, or by interfering with the activity ofone or more desaturases which participate in the catabolism of thesecompounds, makes increasing the yield, production and/or productionefficiency of fatty acid molecules and lipid molecules from organismssuch as microorganisms or plants, possible.

[0083] The mutagenesis of the nucleic acid sequences according to theinvention can give rise to desaturases with modified activities whichindirectly affect the production of one or more desired fine chemicalsfrom microorganisms or plants. For example, desaturases according to theinvention which participate in the export of waste products can exhibita greater number or higher activity, so that the normal metabolic wasteproducts of the cell (whose quantity might be increased owing to theoverproduction of the desired fine chemical) are exported efficientlybefore they can damage the molecules in the cell (which would reducecell viability) or interfere with the biosynthetic pathways of the finechemicals (which would reduce the yield, production or productionefficiency of a desired fine chemical). The relatively largeintracellular amounts of the desired fine chemical themselves canfurthermore be toxic to the cell, so that increasing the activity ornumber of transporters capable of exporting these compounds from thecell results in an increased viability of the cell in culture, which, inturn, leads to a higher number of cells in the culture which produce thedesired fine chemical. The desaturases according to the invention canalso be manipulated in such a way that the corresponding amounts ofdifferent lipid molecules and fatty acid molecules are produced. Thiscan have a substantial effect on the lipid concentration of the cellmembrane. Since each lipid type has different physical properties, amodification of the lipid composition of a membrane can significantlymodify membrane fluidity. Modifications of the membrane fluidity canaffect the transport of molecules via the membrane and cell integrity,each of which has a substantial effect on the production of finechemicals from microorganisms and plants in large-scale fermentationculture. Plant membranes impart specific properties such as tolerance tohigh and low temperatures, salt, drought and tolerance with respect topathogens such as bacteria and fungi. The modulation of the membranecomponents may therefore have a critical effect on the ability of theplants to survive under the abovementioned stress parameters. This cantake place via changes in signal cascades or directly via the modifiedmembrane composition (see, for example, Chapman, 1998, Trends in PlantScience, 3(11):419-426) and signal cascades (see Wang 1999, PlantPhysiology, 120:645-651) or affect the tolerance of low temperatures, asdisclosed in WO 95/18222.

[0084] The isolated nucleic acid sequences according to the inventionare present, for example, in the genome of a Phaeodactylum tricornutumUTEX646 strain which is available via the algae collection of theUniversity of Texas, Austin.

[0085] The nucleotide sequence of the Phaeodactylum tricornutum cDNA andthe derived amino acid sequences of the desaturases are shown in SEQ IDNO: 1 to 6 and 11 and 12. Computer analyses were carried out whichclassify and/or identify these nucleotide sequences as sequences whichencode proteins participating in the metabolism of cell membranecomponents or which participate in the transport of compounds via cellmembranes, or of PUFA biosynthesis. ESTs with the database input NO:PT001070010R and PT001078032R by the inventors constitute the sequencesaccording to the invention in SEQ ID NO: 1 and 3. The sequence of thefragment of EST PT001070010R was determined and is as shown in SEQ IDNO: 5. In a similar manner, the sequence of clone PT001078032R is shownin SEQ ID NO: 1. Gene names were assigned to the clones. Theabbreviations denote: Pp=Physcomitrella patens, Pt=Phaeodactylumtricornutum. PT001070010R of SEQ ID NO: 5 encodes a novel gene which ishomologous to Δ12-desaturase and PT001078032R encodes a novelΔ5-desaturase. Pt_des6 can be isolated in accordance with Example 5a bymeans of polymerase chain reaction with the aid of degenerateoligonucleotides. A fragment obtained in this way can be isolated forscreening a Phaeodactylum tricornutum cDNA library, and the codingregion of a Phaeodactylum tricornutum Δ6-desaturase can be obtained. Agene isolated in this way is termed Pt_des6 in Table 1 and is shown inSEQ ID NO: 3. The corresponding amino acid sequences are obtained bytranslating the genetic code of sequence ID NO: 1, 3 and 5 and aredefined as SEQ ID NO: 2, 4 and 6 (see also Table 1). A further nucleicacid sequence which encodes a Δ12-desaturase can also be found inTable 1. It has the clone number PT001072031R. TABLE 1 Gene Nucleic acidPolypeptide name Clone name SEQ ID NO: SEQ ID NO: Δ5-desaturase Pt_des5PT001078032R 1 2 Δ6-desaturase Pt_des6 Pt_des6 3 4 Δ12-desaturasePt_des12 PT001070010R 5 6 Δ6-desaturase Pp_des6 Pp_des6 7 8 Δ6-elongasePp_PSE1 PP001019019F 9 10 Δ12-desaturase Pt des12.2 PT001072013R 11 12

[0086] The present invention also relates to proteins with an amino acidsequence which is essentially homologous with an amino acid sequence ofSEQ ID NO:2, 4, 6 or 12. As used in the present context, a protein withan amino acid sequence which is essentially homologous with a selectedamino acid sequence has at least approximately 50% homology with theselected amino acid sequence, for example the complete amino acidsequence selected. A protein with an amino acid sequence which isessentially homologous with a selected amino acid sequence can also haveat least approximately 50 to 60% homology, preferably at leastapproximately 60 to 70% homology and more preferably at leastapproximately 70 to 80%, 80 to 90% or 90 to 95% homology and mostpreferably at least approximately 96%, 97%, 98%, 99% or more homologywith a selected amino acid sequence.

[0087] The desaturase according to the invention or the biologicallyactive part or the fragment thereof can participate in the metabolism oflipids required for the synthesis of membranes or storage lipids inmicroorganisms and can, in combination with further genes, in particularthose with elongase activity, contribute to activities required for theelongation of C₁₈- or C₂₀₋₂₂-PUFAs so that C₁₈-, C₂₀-, C₂₂- or C₂₄-PUFAsand related PUFAs are obtained. In this context, desaturases accordingto the invention can be cloned in combination with elongases and otherdesaturases in expression cassettes according to the invention andemployed for the transformation of plants with the aid of Agrobacterium.

[0088] Various aspects of the invention are described in greater detailin the subsections which follow.

[0089] A. Isolated Nucleic Acid Molecules

[0090] One embodiment of the invention are isolated nucleic acidsderived from PUFA-producing microorganisms and encoding polypeptideswhich desaturate C₁₈- or C₂₀₋₂₂-fatty acids with at least one, two,three or four double bonds in the fatty acid.

[0091] A further embodiment according to the invention are isolatednucleic acids encompassing nucleotide sequences encoding polypeptideswhich desaturate C₁₈- or C₂₀-fatty acids with at least one, two, threeor four double bonds in the fatty acid and which are selected from thegroup consisting of

[0092] a) a nucleic acid sequence with the sequence shown in SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11,

[0093] b) nucleic acid sequences which, owing to the degeneracy of thegenetic code, are obtained by backtranslating the amino acid sequencesshown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12,

[0094] c) derivatives of the nucleic acid sequence shown in SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11, which encodepolypeptides with the amino acid sequences shown in SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6 or SEQ ID NO: 12 and have at least 50% homology atthe amino acid level, without essentially reducing the enzymatic actionof the polypeptides.

[0095] The abovementioned nucleic acid according to the invention isderived from organisms such as ciliates, fungi, algae or dinoflagellateswhich are capable of synthesizing PUFAs, preferably from Phaeodactylumtricornutum or closely related organisms.

[0096] One aspect of the invention relates to isolated nucleic acidmolecules which encode desaturase polypeptide or biologically activeparts thereof, and to nucleic acid fragments which suffice for use ashybridization probes or primers for identifying or amplifying adesaturase-encoding nucleic acid (for example desaturase DNA). The term“nucleic acid molecule” as used in the present context is intended toencompass DNA molecules (for example cDNA or genomic DNA) and RNAmolecules (for example mRNA) and DNA or RNA analogs which are generatedby means of nucleotide analogs. This term additionally encompasses theuntranslated sequence on the 3′ and the 5′ ends of the coding generegion: at least 500, preferably 200, especially preferably 100,nucleotides of the sequence upstream of the 5′ end of the coding regionand at least 100, preferably 50, especially preferably 20, nucleotidesof the sequence downstream of the 3′ end of the coding gene region. Thenucleic acid molecule can be single- or double-stranded, but ispreferably double-stranded DNA. An “isolated” nucleic acid molecule isseparated from other nucleic acid molecules which are present in thenatural source of the nucleic acid. An “isolated” nucleic acidpreferably has no sequences which naturally flank the nucleic acid inthe genomic DNA of the organism from which the nucleic acid is derived(for example sequences located at the 5′ and 3′ ends of the nucleicacid). In various embodiments, the isolated desaturase nucleic acidmolecule can comprise, for example, less than approximately 5 kb, 4 kb,3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences whichnaturally flank the nucleic acid molecule in the genomic DNA of the cellfrom which the nucleic acid is derived (for example a Physcomitrellapatens cell). An “isolated” nucleic acid molecule, such as a cDNAmolecule, can moreover be essentially free from other cellular materialor culture medium if it is generated by recombinant techniques, or freefrom chemical precursors or other chemicals if it is synthesizedchemically.

[0097] A nucleic acid molecule according to the invention, for example anucleic acid molecule with a nucleotide sequence of SEQ ID NO:1 or apart thereof, can be isolated using standard techniques of molecularbiology and the sequence information provided herein. Also, for examplea homologous sequence or homologous, conserved sequence regions can beidentified at DNA or amino acid level with the aid of alignmentalgorithms. For example, a Phaeodactylum tricornutum cDNA can beisolated from a Phaeodactylum tricornutum library by using the completeSEQ ID NO:1, 3, 5 or 11 or a part thereof as hybridization probe andstandard hybridization techniques (as described, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Moreover, a nucleic acid moleculeencompassing a complete sequence of SEQ ID NO: 1, 3, 5 or 11 or a partthereof can be isolated by polymerase chain reaction, whereoligonucleotide primers which are generated on the basis of thissequence or parts thereof, in particular regions around motifs ofExample 5a or modifications of the same in individual defined aminoacids are used (for example, a nucleic acid molecule encompassing thecomplete sequence of SEQ ID NO:1, 3, 5 or 11 or a part thereof can beisolated by polymerase chain reaction using oligonucleotide primerswhich have been generated on the basis of this same sequence of SEQ IDNO: 1, 3, 5 or 11). For example, mRNA can be isolated from cells (forexample by the guanidinium thiocyanate extraction method of Chirgwin etal. (1979) Biochemistry 18:5294-5299) and cDNA by means of reversetranscriptase (for example Moloney MLV reverse transcriptase, availablefrom Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase, availablefrom Seikagaku America, Inc., St.Petersburg, Fla.). Syntheticoligonucleotide primers for amplification by means of polymerase chainreaction can be generated on the basis of one of the sequences shown inSEQ ID NO: 1, 3, 5 or 11 and in FIG. 5a or with the aid of the aminoacid sequences shown in SEQ ID NO: 2, 4, 6 or 12. A nucleic acidaccording to the invention can be amplified using cDNA or,alternatively, genomic DNA as template and suitable oligonucleotideprimers, in accordance with standard PCR amplification techniques. Thenucleic acid amplified in this way can be cloned into a suitable vectorand characterized by means of DNA sequence analysis. Oligonucleotideswhich correspond to a desaturase nucleotide sequence can be generated bystandard synthesis methods, for example using an automatic DNAsynthesizer.

[0098] The cDNA shown in SEQ ID NO: 1,3, 5 or 11 encompasses sequenceswhich encode desaturases (i.e. the “coding region”), and 5′-untranslatedsequences and 3′-untranslated sequences. Alternatively, the nucleic acidmolecule can only encompass the coding region of one of the sequences inSEQ ID NO: 1, 3, 5 or 11 or can comprise complete genomic fragmentswhich have been isolated from genomic DNA.

[0099] In a further preferred embodiment, an isolated nucleic acidmolecule according to the invention encompasses a nucleic acid moleculewhich is a complement of one of the nucleotide sequences shown in SEQ IDNO: 1, 3, 5 or 11 or a part thereof. A nucleic acid molecule which iscomplementary to one of the nucleotide sequences shown in SEQ ID NO: 1,3, 5 or 11 is sufficiently complementary if it is capable of hybridizingwith one of the sequences stated in SEQ ID NO: 1, 3, 5 or 11, givingrise to a stable duplex.

[0100] Homologs of the novel desaturase nucleic acid sequences with thesequence SEQ ID NO: 1, 3, 5 or 11 means, for example, allelic variantswith at least approximately 50 to 60% homology, preferably at leastapproximately 60 to 70% homology, more preferably at least approximately70 to 80%, 80 to 90% or 90 to 95% homology and even more preferably atleast approximately 95%, 96% 97%, 98%, 99% or more homology with one ofthe nucleotide sequences shown in SEQ ID NO: 1, 3, 5 or 11 or theirhomologs, derivatives, analogs or parts thereof. In a further preferredembodiment, an isolated nucleic acid molecule according to the inventionencompasses a nucleotide sequences which hybridizes with one of thenucleotide sequences shown in SEQ ID NO: 1, 3, 5 or 11 or a partthereof, for example under stringent conditions. Allelic variantsencompass, in particular, functional variants which can be obtained bythe deletion, insertion or substitution of nucleotides from/into thesequence shown in SEQ ID NO: 1, 3, 5 or 11, it being intended, however,for the enzyme activity of the resulting proteins which are synthesizedto be advantageously retained for the insertion of one or more genes.Proteins which retain the enzymatic activity of desaturase, that is tosay whose activity is essentially not reduced, means proteins with atleast 10%, preferably 20%, especially preferably 30%, very particularlypreferably 40%, of the original enzyme activity compared with theprotein encoded by SEQ ID NO: 2, 4, 6 or 12.

[0101] Homologs of SEQ ID NO: 1, 3, 5 or 11 also means, for example,bacterial, fungal and plant homologs, truncated sequences,single-stranded DNA or RNA of the coding and noncoding DNA sequence.

[0102] Homologs of SEQ ID NO: 1, 3, 5 or 11 also means derivatives suchas, for exmaple, promoter variants. The promoters upstream of thenucleotide sequences stated can be modified by one or more nucleotidesubstitutions, by insertion(s) and/or deletion(s), without, however,interfering with the functionality or activity of the promoters. It isfurthermore possible for the activity of the promoters to be increasedby modifying their sequence or for them to be replaced completely bymore active promoters, even from heterologous organisms.

[0103] Moreover, the nucleic acid molecule according to the inventionmay only encompass part of the coding region of one of the sequences inSEQ ID NO: 1, 3, 5 or 11, for example a fragment which can be used asprobe or primer, or a fragment which encodes a biologically activesegment of a desaturase. The nucleotide sequences determined fromcloning the Phaeodactylum tricornutum desaturase gene allow thegeneration of probes and primers which are designed for identifyingand/or cloning desaturase homologs in other cell types and organisms anddesaturase homologs from other microalgae or related species. Theprobe/primer usually encompasses an essentially purifiedoligonucleotide. The oligonucleotide usually encompasses a nucleotidesequence region which hybridizes under stringent conditions to at leastapproximately 12, preferably approximately 16, more preferablyapproximately 25, 40, 50 or 75 successive nucleotides of a sense strandof one of the sequences stated in SEQ ID NO: 1, 3, 5 or 11, of anantisense strand of one of the sequences stated in SEQ ID NO: 1, 3, 5 or11 or its homologs, derivatives or analogs or naturally occurringmutants thereof. Primers based on a nucleoide sequence of SEQ ID NO: 1,3, 5 or 11 can be used in PCR reactions for cloning desaturase homologs.Probes based on the desaturase nucleotide sequences can be used fordetecting transcripts or genomic sequences which encode the same orhomologous proteins. In preferred embodiments, the probe additionallyencompasses a labeling group bound thereto, for example a radioisotope,a fluorescent compound, an enzyme or an enzyme cofactor. These probescan be used as part of a test kit for genomic markers for identifyingcells which misexpress a desaturase, for example by measuring an amountof a desaturase-encoding nucleic acid in a cell sample, for examplemeasuring the desaturase mRNA level, or for determining whether agenomic desaturase gene is mutated or deleted.

[0104] In one embodiment, the nucleic acid molecule according to theinvention encodes a protein or part thereof which encompasses an aminoacid sequence with sufficient homology with an amino acid sequence ofSEQ ID NO: 2, 4, 6 or 12 for the protein or part thereof to retain theability to participate in the metabolism of compounds required for thesynthesis of the cell membranes in microorganisms or plants or in thetransport of molecules via these membranes. As used in the presentcontext, the term “sufficient homology” refers to proteins or partsthereof whose amino acid sequences have a minimum number of amino acidresidues which are identical with or equivalent to an amino acidsequence of SEQ ID NO:2 (for example an amino acid residue with asimilar side chain, such as an amino acid residue in one of thesequences of SEQ ID NO:2) so that the protein or the part thereof canparticipate in the metabolism of compounds required for the synthesis ofcell membranes in microorganisms or plants or in the transport ofmolecules via these membranes. As described herein, protein componentsof these metabolic pathways for membrane components or membranetransport systems can play a role in the production and secretion of oneor more fine chemicals. Examples of these activities are also describedherein. Thus, the “function of a desaturase” contributes either directlyor indirectly to the yield, production and/or production efficiency ofone or more fine chemicals. Examples of desaturase substrate specificityof the catalytic activity are stated in Tables 5 and 6.

[0105] In a further embodiment, derivatives of the nucleic acid moleculeaccording to the invention encode proteins with at least approximately50 to 60% homology, preferably at least approximately 60 to 70% homologyand more preferably at least approximately 70 to 80%, 80 to 90%, 90 to95% homology, and most preferably at least approximately 96%, 97%, 98%,99% or more homology with a complete amino acid sequence of SEQ ID NO:2.The homology of the amino acid sequence can be determined over theentire sequence region using the program PileUp (J. Mol. Evolution., 25,351-360, 1987, Higgins et al., CABIOS, 5, 1989:151-153) or BESTFIT orGAP (Henikoff, S. and Henikoff, J. G. (1992). Amino acid substitutionmatrices from protein blocks. Proc. Natl. Acad. Sci. USA 89:10915-10919.)

[0106] Parts of proteins encoded by the desaturase nucleic acidmolecules according to the invention are preferably biologically activeparts of one of the desaturases. As used herein, the term “biologicallyactive part of a desaturase” is intended to encompass a segment, forexample a domain/motif, of a desaturase which can participate in themetabolism of compounds required for the synthesis of cell membranes inmicroorganisms or plants or in the transport of molecules via thesemembranes or which has an activity stated in Tables 5 and 6. An assay ofthe enzymatic activity can be carried out in order to determine whethera desaturase or a biologically active part thereof can participate inthe metabolism of compounds required for the synthesis of cell membranesin microorganisms or plants or in the transport of molecules via thesemembranes. These assay methods as described in detail in Example 8 ofthe examples section are known to the skilled worker.

[0107] Additional nucleic acid fragments which encode biologicallyactive segments of a desaturase can be generated by isolating part ofone of the sequences in SEQ ID NO: 1, 3, 5 or 11, expressing the encodedsegment of the desaturase or of the peptide (for example by recombinantexpression in vitro) and determining the activity of the encoded part ofthe desaturase or of the peptide.

[0108] Moreover, the invention encompasses nucleic acid molecules whichdiffer from one of the nucleotide sequences shown in SEQ ID NO: 1, 3, 5or 11 (and parts thereof) owing to the degeneracy of the genetic codeand which thus encode the same desaturase as the one encoded by thenucleotide sequences shown in SEQ ID NO: 1, 3, 5 or 11. In anotherembodiment, an isolated nucleic acid molecule according to the inventionhas a nucleotide sequence which encodes a protein with an amino acidsequence shown in SEQ ID NO: 2, 4, 6 or 12. In a further embodiment, thenucleic acid molecule according to the invention encodes a full-lengthdesaturase protein which is essentially homologous to an amino acidsequence of SEQ ID NO: 2, 4, 6 or 12 (which is encoded by an openreading frame shown in SEQ ID NO: 1, 3, 5 or 11) and which can beidentified and isolated by customary methods.

[0109] In addition to the desaturase nucleotide sequence shown in SEQ IDNO: 1, 3, 5 or 11, the skilled worker recognizes that DNA sequencepolymorphisms may exist which lead to changes in the amino acidsequences of the desaturases within a population (for example thePhaeodactylum tricornutum population). These genetic polymorphisms inthe desaturase gene can exist between individuals within a populationowing to natural variation. As used in the present context, the terms“gene” and “recombinant gene” refer to nucleic acid molecules with anopen reading frame which encodes a desaturase, preferably aPhaeodactylum tricornutum desaturase. These natural variants usuallycause a variance of 1 to 5% in the nucleotide sequence of the desaturasegene. All of these nucleotide variations and resulting amino acidpolymorphisms in desaturase which are the result of natural variationand do not alter the functional activity of desaturases are intended tocome within the scope of the invention.

[0110] Nucleic acid molecules which correspond to the natural variantsand non-Phaeodactylum-tricornutum-homologs, -derivatives and -analogs ofthe Phaeodactylum tricornutum cDNA can be isolated in accordance withstandard hybridization techniques under stringent hybridizationconditions owing to their homology with the Phaeodactylum tricornutumdesaturase nucleic acid disclosed herein using the Phaeodactylumtricornutum cDNA or part thereof as hybridization probe. In anotherembodiment, an isolated nucleic acid molecule according to the inventionhas a minimum length of 15 nucleotides and hybridizes under stringentconditions to the nucleic acid molecule which encompasses a nucleotidesequence of SEQ ID NO:1, 3, 5 or 11.

[0111] In other embodiments, the nucleic acid has a minimum length of25, 50, 100, 250 or more nucleotides. The term “hybridizes understringent conditions” as used in the present context is intended todescribe hybridization and wash conditions under which nucleotidesequences which have at least 60% homology to each other usually remainhybridized to each other. The conditions are preferably such thatsequences which have at least approximately 65% homology, morepreferably approximately 70% homology and even more preferably at leastapproximately 75% or more homology to each other usually remainhybridized to each other. These stringent conditions are known to theskilled worker and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. A preferred,nonlimiting example of stringent hybridization conditions arehybridizations in 6×sodium chloride/sodium citrate (=SSC) atapproximately 45° C., followed by one or more wash steps in 0.2×SSC,0.1% SDS at 50 to 65° C. It is known to the skilled worker that thesehybridization conditions differ depending on the type of nucleic acidand, for example, when organic solvents are present, with regard to thetemperature and the concentration of the buffer. The temperaturediffers, for example, under “standard hybridization conditions”depending on the type of the nucleic acid between 42° C. and 58° C. inaqueous buffer with a concentration of 0.1 to 5×SSC (pH 7.2). If organicsolvent is present in the abovementioned buffer, for example 50%formamide, the temperature under standard conditions is approximately42° C. The hybridization conditions for DNA:DNA hybrids are preferablyfor example 0.1×SSC and 20° C. to 45° C., preferably between 30° C. and45° C. The hybridization conditions for DNA:RNA hybrids are preferablyfor example 0.1×SSC and 30° C. to 55° C., preferably between 45° C. and55° C. The abovementioned hybridization temperatures are determined forexample for a nucleic acid approximately 100 bp (=base pairs) in lengthand a G+C content of 50% in the absence of formamide. The skilled workerknows how the hybridization conditions required can be determined withreference to textbooks, such as the one mentioned above, or from thefollowing textbooks: Sambrook et al., “Molecular Cloning”, Cold SpringHarbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, “Nucleic AcidsHybridization: A Practical Approach”, IRL Press at Oxford UniversityPress, Oxford; Brown (Ed.) 1991, “Essential Molecular Biology: APractical Approach”, IRL Press at Oxford University Press.

[0112] Preferably, an isolated nucleic acid molecule according to theinvention which hybridizes under stringent conditions to a sequence ofSEQ ID NO:1, 3, 5 or 11 corresponds to a naturally occurring nucleicacid molecule. As used in the present context, a “naturally occurring”nucleic acid molecule refers to an RNA or DNA molecule with a nucleotidesequence which occurs in nature (for example which encodes a naturalprotein). In one embodiment, the nucleic acid encodes a naturallyoccurring Phaeodactylum tricornutum desaturase.

[0113] In addition to naturally occurring variants of the desaturasesequence which may exist in the population, the skilled workerfurthermore recognizes that changes by means of mutation may also beintroduced into a nucleotide sequence of SEQ ID NO: 1, 3, 5 or 11, whichleads to changes in the amino acid sequence of the encoded desaturasewithout adversely affecting the functionality of the desaturase protein.For example, nucleotide substitutions which lead to amino acidsubstitutions on “nonessential” amino acid residues can be generated ina sequence of SEQ ID NO: 2, 4, 6 or 12. A “nonessential” amino acidresidue is a residue which can be altered in a wild-type sequence of oneof the desaturases (SEQ ID NO: 2, 4, 6 or 12) without altering, that isto say essentially reducing, the activity of the desaturase, while an“essential” amino acid residue is required for the desaturase activity.Other amino acid residues (for example those which are not conserved, oronly semi-conserved, in the domain with desaturase activity), however,may not be essential for the activity and can therefore be modifiedwithout modifying the desaturase activity.

[0114] Accordingly, a further aspect of the invention relates to nucleicacid molecules which encode desaturases comprising modified amino acidresidues which are not essential for the desaturase activity. Thesedesaturases differ from a sequence in SEQ ID NO: 2, 4, 6 or 12 withregard to the amino acid sequence while still retaining at least one ofthe desaturase activities described herein. In one embodiment, theisolated nucleic acid molecule encompasses a nucleotide sequenceencoding a protein, the protein encompassing an amino acid sequence withat least approximately 50% homology with an amino acid sequence of SEQID NO: 2, 4, 6 or 12 and being able to participate in the metabolism ofcompounds required for the synthesis of the cell membranes inPhaeodactylum tricornutum or in the transport of molecules via thesemembranes. The protein encoded by the nucleic acid molecule preferablyhas at least approximately 50 to 60% homology with one of the sequencesin SEQ ID NO:2, 4, 6 or 12, more preferably at least approximately 60 to70% homology with one of the sequences in SEQ ID NO:2, 4, 6 or 12, evenmore preferably at least approximately 70 to 80%, 80 to 90%, 90 to 95%homology with one of the sequences in SEQ ID NO: 2, 4, 6 or 12 and mostpreferably at least 96%, 97%, 98% or 99% homology with one of thesequences in SEQ ID NO: 2, 4, 6 or 12.

[0115] To determine the percentage homology of two amino acid sequences(for example one of the sequences of SEQ ID NO: 2, 4, 6 or 12 and amutated form thereof) or of two nucleic acids, the sequences are writtenone underneath the other to allow optimum comparison (for example, gapsmay be introduced into the sequence of a protein or of a nucleic acid inorder to generate an optimal alignment with the other protein or theother nucleic acid). Then, the amino acid residues or nucleotides at thecorresponding amino acid positions or nucleotide positions are compared.If a position in a sequence (for example one of the sequences of SEQ IDNO: 2, 4, 6 or 12) is occupied by the same amino acid residue or thesame nucleotide as the corresponding position in the other sequence (forexample a mutated form of the sequence selected from SEQ ID NO: 2, 4, 6or 12), then the molecules are homologous at this position (i.e. aminoacid or nucleic acid “homology” as used in the present contextcorresponds to amino acid or nucleic acid “identity”). The percentagehomology between the two sequences is a function of the number ofidentical positions which the sequences share (i.e. % homology=number ofidentical positions/total number of positions×100). The terms homologyand identity are thus to be considered as being synonymous.

[0116] An isolated nucleic acid molecule which encodes a desaturasewhich is homologous with a protein sequence of SEQ ID NO: 2, 4, 6 or 12can be generated by introducing one or more nucleotide substitutions,additions or deletions into a nucleotide sequence of SEQ ID NO: 1, 3, 5or 11 so that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced into one of the sequences of SEQ ID NO: 1, 3, 5 or 11 bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions aregenerated at one or more of the predicted nonessential amino acidresidues. In a “conservative amino acid substitution”, the amino acidresidue is exchanged for an amino acid residue with a similar sidechain. Families of amino acid residues with similar side chains havebeen defined in the specialist field. These families encompass aminoacids with basic side chains (for example lysine, arginine, hystidine),acidic side chains (for example aspartic acid, glutamic acid), unchargedpolar side chains (for example glycine, asparagine, glutamine, serine,threonine, tyrosine, cysteine), unpolar side chains (for examplealanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (for examplethreonine, valine, isoleucine) and aromatic side chains (for exampletyrosine, phenylalanine, tryptophan, histidine). A predictednonessential amino acid residue in a desaturase is thus preferablyexchanged for another amino acid residue from the same side-chainfamily. As an alternative, in another embodiment, the mutations can beintroduced randomly over all or part of the desaturase-encodingsequence, for example by saturation mutagenesis, and the resultingmutants can be screened for the desaturase activity in order to identifymutants which retain desaturase activity. Following the mutagenesis ofone of the sequences of SEQ ID NO: 1, 3, 5 or 11, the encoded proteincan be expressed recombinantly, and the activity of the protein can bedetermined, for example using the assays described herein (see examplessection).

[0117] In addition to the nucleic acid molecules which encode theabove-described desaturases, a further aspect of the invention relatesto isolated nucleic acid molecules which are “antisense” to the nucleicacid sequences according to the invention. An “antisense” nucleic acidencompasses a nucleotide sequence which is complementary to a “sense”nucleic acid which encodes a protein, for example complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can bind to asense nucleic acid by hydrogen bonds. The antisense nucleic acid can becomplementary to a complete desaturase-encoding strand or only to partthereof. In one embodiment, an antisense nucleotide acid molecule is“antisense” to a “coding region” of the coding strand of a nucleotidesequence encoding a desaturase. The term “coding region” refers to theregion of the nucleotide sequence which encompasses codons which aretranslated into amino acid residues (for example the entire codingregion which starts and ends with the stop codon, i.e. the last codonbefore the stop codon). In a further embodiment, the antisense nucleicacid molecule is “antisense” to a “noncoding region” of the codingstrand of a nucleotide sequence encoding desaturase. The term “noncodingregion” refers to 5′ and 3′ sequences which flank the coding region andare not translated into amino acids (i.e. which are also termed 5′- and3′-untranslated regions).

[0118] Given the desaturase-encoding sequences disclosed herein of thecoding strand (for example the sequences shown in SEQ ID NO: 1, 3, 5 or11), antisense nucleic acids according to the invention can be designedin accordance with the rules of Watson-Crick base pairing. The antisensenucleic acid molecule can be complementary to all of the coding regionof desaturase mRNA, but is more preferably an oligonucleotide which is“antisense” to only part of the coding or noncoding region of thedesaturase mRNA. The antisense oligonucleotide can be complementary, forexample, to the region around the translation start of desaturase mRNA.An antisense oligonucleotide can have a length of, for example,approximately 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 and morenucleotides. An antisense nucleic acid according to the invention can beconstructed by processes known in the art using chemical synthesis andenzymatic ligation reactions. An antisense nucleic acid (for example anantisense oligonucleotide) can, for example, be synthesized chemically,making use of naturally occurring nucleotides or variously modifiednucleotides which are such that they increase the biological stabilityof the molecules or increase the physical stability of the duplex formedbetween the antisense and the sense nucleic acid; for example,phosphorothioate derivatives and acridine-substituted nucleotides may beused. Examples of modified nucleotides which may be used for generatingthe antisense nucleic acid are, inter alia, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentyladenine, uracil-5-oxyacetic acid (v),wybutoxosin, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)wand 2,6-diaminopurine. The antisense nucleic acid can, alternatively, begenerated biologically using an expression vector into which a nucleicacid has been subcloned in antisense orientation (i.e. RNA which istranscribed by the nucleic acid introduced is in antisense orientationrelative to a target nucleic acid of interest, which is described ingreater detail in the subsection which follows).

[0119] The antisense nucleic acid molecules according to the inventionare usually administered to a cell or generated in situ so that theyhybridize with, or bind to, the cellular mRNA and/or the genomic DNAencoding a desaturase, thus inhibiting expression of the protein, forexample by inhibiting transcription and/or translation. Hybridizationcan be effected by conventional nucleotide complementarity with theformation of a stable duplex or, for example in the case of an antisensenucleic acid molecule which binds DNA duplices, by specific interactionsin the major groove of the double helix. The antisense molecule can bemodified in such a manner that it specifically binds to a receptor or toan antigen expressed at the selected cell surface, for example bybinding the antisense nucleic acid molecule to a peptide or an antibody,each of which binds to a cell surface receptor or an antigen. The cellscan also be provided with the antisense nucleic acid molecule using thevectors described herein. Vector constructs in which the antisensenucleic acid molecule is under the control of a strong prokaryotic,viral or eukaryotic promoter, including a plant promoter, are preferredfor achieving sufficient intracellular concentrations of the antisensemolecules.

[0120] In a further embodiment, the antisense nucleic acid moleculeaccording to the invention is an α-anomeric nucleic acid molecule. Anα-anomeric nucleic acid molecule forms specific double-stranded hybridswith complementary RNA, the strands running parallel to each other, incontrast to ordinary β units (Gaultier et al. (1987) Nucleic Acids Res.15:6625-6641). Moreover, the antisense nucleic acid molecule canencompass a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic AcidsRes. 15:6131-6148) or a chimeric RNA-DNA analog (Inoue et al. (1987)FEBS Lett. 215:327-330).

[0121] In a further embodiment, an antisense nucleic acid according tothe invention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which can cleave a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (for example hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used for the catalyticcleavage of desaturase-mRNA transcripts, in order thereby to inhibit thetranslation of desaturase mRNA. A ribozyme with specificity for adesaturase-encoding nucleic acid can be designed on the basis of thenucleotide sequence of one of the desaturase-cDNAs disclosed in SEQ IDNO: 1, 3, 5 or 11 (i.e. or on the basis of a heterologous sequence to beisolated in accordance with the methods taught in the presentinvention). For example, a derivative of a Tetrahymena-L-19-IVS RNA canbe constructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in adesaturase-encoding mRNA. See, for example, Cech et al., U.S. Pat. No.4,987,071 and Cech et al., U.S. Pat. No. 5,116,742. As an alternative,desaturase mRNA can be used for selecting a catalytic RNA with aspecific ribonuclease activity from among a pool of RNA molecules. See,for example, Bartel, D., and Szostak, J. W. (1993) Science261:1411-1418.

[0122] As an alternative, desaturase gene expression can be inhibited bydirecting nucleotide sequences which are complementary to the regulatoryregion of a desaturase nucleotide sequence (for example a desaturasepromoter and/or enhancer) in such a way that triple helix structures areformed which inhibit the transcription of a desaturase gene in targetcells. See, in general, Helene, C. (1991) Anticancer Drug Res. 6(6)569-84; Helene, C., et al. (1992) Ann. N. Y. Acad. Sci. 660:27-36; andMaher. L. J. (1992) Bioassays 14(12):807-815.

[0123] B. Gene Construct (=Nucleic Acid Construct, Nucleic Acid Fragmentor Expression Cassette)

[0124] The expression cassette according to the invention is to beunderstood as meaning the sequences mentioned in SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5 or SEQ ID NO: 11 which are the result of the geneticcode, and/or their functional or nonfunctional derivatives, which wereadvantageously linked functionally to one or more regulatory signals forincreasing gene expression and which advantageously control theexpression of the coding sequence in the host cell. These regulatorysequences are intended to make possible the targeted expression of thegenes and the protein expression. Depending on the host organism, thismay mean, for example, that the gene is expressed and/or overexpressedonly after induction or else that it is expressed and/or overexpressedimmediately. For example, these regulatory sequences take the form ofsequences to which inductors or repressors bind, thus regulating theexpression of the nucleic acid. In addition to these novel regulatorysequences, or instead of these sequences, the natural regulation ofthese sequences may still be present before the actual structural genesand, if appropriate, may have been modified genetically, so that naturalregulation was eliminated and the expression of the genes increased.However, the gene construct may also have a simpler structure, that isto say no additional regulatory signals have been inserted before thenucleic acid sequence or its derivatives, and the natural promotertogether with its regulation has not been removed. Instead, the naturalregulatory sequence has been mutated in such a way that regulation nolonger takes place and/or gene expression is increased. These modifiedpromoters may also be arranged by themselves in the form ofpart-sequences (=promoter with parts of the nucleic acid sequencesaccording to the invention) before the natural gene in order to increasethe activity. Moreover, the gene construct may advantageously alsocomprise one or more of what are known as enhancer sequences linkedfunctionally to the promoter, and these make possible an increasedexpression of the nucleic acid. It is also possible to insert additionaladvantageous sequences on the 3′ end of the DNA sequences, such asfurther regulatory elements or terminators. TheΔ5-desaturase/Δ6-desaturase and/or Δ12-desaturase genes may be presentin one or more copies in the expression cassette (=gene construct).

[0125] In this context, the regulatory sequences or factors canpreferably have a positive effect on, and thus increase, the expressionof the genes introduced, as has been described above. An enhancement ofthe regulatory elements can advantageously take place at thetranscriptional level by using strong transcription signals such aspromoters and/or enhancers. In addition, however, translation may alsobe enhanced, for example by increasing the stability of the mRNA.

[0126] A further embodiment of the invention are one or more geneconstructs comprising one or more sequences which are defined by SEQ IDNO: 1, 3, 5, 7, 9 or 11 and which encode polypeptides in accordance withSEQ ID NO: 2, 4, 6, 8, 10 or 12. SEQ ID NO: 1, 3, 5, 7 and 11 arederived from desaturases, while SEQ ID NO: 9 encodes an elongase.Desaturases encode enzymes which introduce a double bond at the Δ5, Δ6or Δ12 position, the substrate having one, two, three or four doublebonds. The sequence shown in SEQ ID NO: 9 encodes an enzyme activitywhich elongates a fatty acid by at least two carbon atoms, and thehomologs, derivatives or analogs which are linked functionally to one ormore regulatory signals, advantageously for increasing gene expression.Examples of these regulatory sequences are sequences to which inductorsor repressors bind, thus regulating the expression of the nucleic acid.In addition to these novel regulatory sequences, the natural regulationof these sequences may still be present before the actual structuralgenes and, if appropriate, can have been genetically modified so thatthe natural regulation has been eliminated and the expression of thegenes has been increased. However, the gene construct may also have asimpler structure, that is to say no additional regulatory signals havebeen inserted before the sequence SEQ ID NO: 1, 3, 5 or 11 or theirhomologs and the natural promoter with its regulation has not beendeleted. Instead, the natural regulatory sequence has been mutated insuch a way that regulation no longer takes place and gene expression isenhanced. The gene construct may furthermore advantageously encompassone or more of what are known as enhancer sequences which are linkedfunctionally to the promoter and which make possible increasedexpression of the nucleic acid sequence. It is also possibleadditionally to insert advantageous sequences at the 3′ end of the DNAsequences, for example further regulatory elements or terminators. Thedesaturase genes and the elongase gene may be present in one or morecopies in the gene construct. They may be present in one gene constructor more than one gene construct. This gene construct or the geneconstructs can be expressed together in the host organism. In thiscontext, the gene construct or the gene constructs can be inserted intoone or more vectors and be present in the cell in free form or elseinserted into the genome. It is advantageous for the insertion offurther genes into organisms if further genes are present in the geneconstruct.

[0127] Advantageous regulatory sequences for the novel process exist,for example, in promoters such as the cos, tac, trp, tet, trp-tet, lpp,lac, lpp-lac, lacI^(q−), T7, T5, T3, gal, trc, ara, SP6, λ-P_(R) orλ-P_(L) promoter and are advantageously used in Gram-negative bacteria.Further advantageous regulatory sequences exist, for example, in theGram-positive promoters amy and SPO2, in the yeast or fungal promotersADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plantpromoters CaMV 35S [Franck et al., Cell 21 (1980) 285-294], PRP1 [Wardet al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33,nos or in the ubiquitin or phaseolin promoter. Advantageous in thiscontext are also inducible promoters, such as the promoters described inEP-A-0 388 186 (benzylsulfonamide-inducible), Plant J. 2, 1992:397-404(Gatz et al., tetracyclin-inducible), EP-A-0 335 528(abscisic-acid-inducible) or WO 93/21334 (ethanol- orcyclohexenol-inducible). Further suitable plant promoters are thepromoter of cytosolic FBPase or the potato ST-LSI promoter (Stockhaus etal., EMBO J. 8, 1989, 2445), the Glycine max phosphoribosylpyrophosphateamidotransferase promoter (Genbank Accession No. U87999) or thenode-specific promoter described in EP-A-0 249 676. Especiallyadvantageous promoters are those which allow expression in tissues whichare involved in fatty acid biosynthesis. Very especially advantageousare seed-specific promoters such as the USP promoter in accordance withthe embodiment, and also other promoters such as the LEB4 (Baeumlein etal., Plant J., 1992, 2 (2):233-239), DC3 (Thomas, Plant Cell 1996,263:359-368), the phaseolin or the napin promotor. Further especiallyadvantageous promoters are seed-specific promoters which can be used formonocots or dicots which are described in U.S. Pat. No. 5,608,152(oilseed rape napin promoter), WO 98/45461 (Arabidopsis oleosinpromoter), U.S. Pat. No. 5,504,200 (Phaseolus vulgaris phaseolinpromoter), WO 91/13980 (Brassica Bce4 promoter), by Baeumlein et al.,Plant J., 1992, 2 (2):233-239 (LeB4 promoter from a legume), thesepromoters being suitable for dicots. The following promoters aresuitable, for example, for monocots: the barley lpt-2 or lpt-1 promoter(WO 95/15389 and WO 95/23230), the barley Hordein promoter, and othersuitable promoters described in WO 99/16890.

[0128] In principle, it is possible to use all natural promoters withtheir regulatory sequences, such as those mentioned above, for the novelprocess. It is also possible and advantageous additionally to usesynthetic promoters.

[0129] As described above, the gene construct can also encompass furthergenes which are to be introduced into the organisms. It is possible andadvantageous to introduce into the host organisms, and to expresstherein, regulatory genes such as genes for inductors, repressors orenzymes which, owing to the enzymatic activity, engage in the regulationof one or more genes of a biosynthetic pathway. These genes can be ofheterologous or homologous origin. Moreover, the nucleic acid constructor gene construct may advantageously comprise further biosynthesis genesof the fatty acid or lipid metabolism or else these genes may be presenton a further, or several further, nucleic acid constructs. Abiosynthesis gene of the fatty acid or lipid metabolism which isadvantageously selected is a gene from the group consisting of acyl-CoAdehydrogenase(s), acyl-ACP[=acyl carrier protein] desaturase(s),acyl-ACP thioesterase(s), fatty acid acyltransferase(s), fatty acidsynthase(s), fatty acid hydroxylase(s), acetyl-coenzyme Acarboxylase(s), acyl-coenzyme, A-oxidase(s), fatty acid desaturase(s),fatty acid acetylenases, lipoxygenases, triacylglycerol lipases,allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s) ortheir combinations.

[0130] For expressing the other genes which are present, gene constructsadvantageously encompass further 3′- and/or 5′-terminal regulatorysequences for enhancing expression, and these are selected for optimalexpression as a function of the host organism chosen and the gene(s).These regulatory sequences, as mentioned above, are intended to makepossible the specific expression of the genes and protein expression.Depending on the host organism, this may mean, for example, that thegene is expressed or overexpressed only after induction, or that it isexpressed and/or overexpressed immediately.

[0131] Moreover, the regulatory sequences or regulatory factors canpreferably have an advantageous effect on the expression of the geneswhich have been introduced, thus enhancing them. In this manner, it ispossible that the regulatory elements are advantageously enhanced at thetranscriptional level, using strong transcription signals such aspromoters and/or enhancers. However, it is furthermore also possible toenhance translation, for example by improving mRNA stability.

[0132] C. Recombinant Expression Vectors and Host Cells

[0133] A further aspect of the invention relates to vectors, preferablyexpression vectors, comprising a nucleic acid encoding a desaturasealone (or a part thereof) or a nucleic acid construct described underitem B in which the nucleic acid according to the invention is presentalone or in combination with further biosynthesis genes of the fattyacid or lipid metabolism, such as desaturases or elongases. As used inthe present context, the term “vector” refers to a nucleic acid moleculewhich can transport another nucleic acid to which it is bound. One typeof vector is a “plasmid”, which represents a circular double-strandedDNA loop into which additional DNA segments can be ligated. A furthertype of vector is a viral vector, it being possible for additional DNAsegments to be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they havebeen introduced (for example bacterial vectors with a bacterial originof replication, and episomal mammalian vectors). Other vectors (forexample nonepisomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell and are thus replicatedtogether with the host genome. In addition, certain vectors can governthe expression of genes to which they are linked functionally. Thesevectors are referred to as “expression vectors” herein. Usually,expression vectors which are suitable for recombinant DNA techniques cantake the form of plasmids. In the present description, “plasmid” and“vector” may be used interchangeably since the plasmid is the mostfrequently used form of vector. However, the invention is intended toencompass these other forms of expression vectors, such as viral vectors(for example replication-deficient retroviruses, adenoviruses andadeno-related viruses) which exert similar functions. Furthermore, theterm vector is also intended to encompass other vectors known to theskilled worker, such as phages, viruses such as SV40, CMV, baculovirus,adenovirus, transposons, IS elements, phasmids, phagemids, cosmids,linear or circular DNA.

[0134] The recombinant expression vectors according to the inventionencompass a nucleic acid according to the invention or a gene constructaccording to the invention in a form which is suitable for expressingthe nucleic acid in a host cell, which means that the recombinantexpression vectors encompass one or more regulatory sequences, selectedon the basis of the host cells to be used for expression, which is/arelinked functionally to the nucleic acid sequence to be expressed. In arecombinant expression vector “linked functionally” means that thenucleotide sequence of interest is bound to the regulatory sequence(s)in such a way that expression of the nucleotide sequence is possible andthat they are bound to each other so that both sequences fulfil thepredicted function which has been ascribed to the sequence (for examplein an in-vitro transcription/translation system or in a host cell, whenthe vector is introduced into the host cell). The term “regulatorysequence” is intended to encompass promoters, enhancers and otherexpression control elements (for example polyadenylation signals). Theseregulatory sequences are described, for example, in Goeddel: GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990), or see: Gruber and Crosby, in: Methods in PlantMolecular Biology and Biotechnolgy, CRC Press, Boca Raton, Fla., Ed.:Glick and Thompson, Chapter 7, 89-108, including the references therein.Regulatory sequences encompass those which control the constitutiveexpression of a nucleotide sequence in many types of host cell and thosewhich control the direct expression of the nucleotide sequence only incertain host cells under certain conditions. The skilled worker knowsthat the design of the expression vector may depend on factors such asthe choice of the host cell to be transformed, the extent to which thedesired protein is expressed, and the like. The expression vectorsaccording to the invention can be introduced into host cells in order toproduce proteins or peptides, including fusion proteins or fusionpeptides, which are encoded by the nucleic acids as described herein(for example desaturases, mutant forms of desaturases, fusion proteinsand the like).

[0135] The recombinant expression vectors according to the invention canbe designed for expressing desaturases and elongases in prokaryotic andeukaryotic cells. For example, desaturase genes can be expressed inbacterial cells, such as C. glutamicum, insect cells (using baculovirusexpression vectors), yeast and other fungal cells (see Romanos, M. A.,et al. (1992) “Foreign gene expression in yeast: a review”, Yeast8:423-488; van den Hondel, C. A. M. J. J., et al. (1991) “Heterologousgene expression in filamentous fungi”, in: More Gene Manipulations inFungi, J. W. Bennet & L. L. Lasure, Ed., pp. 396-428: Academic Press:San Diego; and van den Hondel, C. A. M. J. J., & Punt, P. J. (1991)“Gene transfer systems and vector development for filamentous fungi”,in: Applied Molecular Genetics of Fungi, Peberdy, J. F., et al., Ed.,pp. 1-28, Cambridge University Press: Cambridge), algae (Falciatore etal., 1999, Marine Biotechnology.1, 3:239-251), ciliates of the followingtypes: Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena,Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella and Stylonychia, inparticular the species Stylonychia lemnae, using vectors and following atransformation method as described in WO 98/01572, and cells ofmulticelled plants (see Schmidt, R. and Willmitzer, L. (1988) “Highefficiency Agrobacterium tumefaciens-mediated transformation ofArabidopsis thaliana leaf and cotyledon explants” Plant CellRep.:583-586; Plant Molecular Biology and Biotechnology, C Press, BocaRaton, Fla., Chapter 6/7, pp. 71-119 (1993); F. F. White, B. Jenes etal., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press (1993),128-43; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42(1991), 205-225 (and references cited therein)) or mammalian cells.Suitable host cells are furthermore discussed in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). As an alternative, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

[0136] In prokaryotes, proteins are usually expressed with vectorscontaining constitutive or inducible promoters which control theexpression of fusion proteins or nonfusion proteins. Fusion vectors adda series of amino acids to a protein encoded therein, usually on theamino terminus of the recombinant protein, but also on the C terminus orfused within suitable regions in the proteins. These fusion vectorsusually have three tasks: 1) to enhance the expression of recombinantprotein; 2) to increase the solubility of the recombinant protein and 3)to support the purification of the recombinant protein by acting asligand in affinity purification. In the case of fusion expressionvectors, a proteolytic cleavage site is frequently introduced at thesite where the fusion moiety and the recombinant protein are linked, sothat the recombinant protein can be separated from the fusion unit afterpurification of the fusion protein. These enzymes and theircorresponding recognition sequences encompass factor Xa, thrombin andenterokinase.

[0137] Typical fusion expression vectors are, inter alia, PGEX(Pharmacia Biotech Inc; Smith, D. B., and Johnson, K. S. (1988) Gene67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.), where glutathione S-transferase (GST),maltose-E-binding protein or protein A is fused to the recombinanttarget protein. In one embodiment, the desaturase-encoding sequence iscloned into a pGEX expression vector to generate a vector encoding afusion protein which encompasses, from the N terminus to the C terminus,GST-thrombin cleavage site-X-protein. The fusion protein can be purifiedby affinity chromatography using glutathione-agarose resin.

[0138] Recombinant desaturase which is not fused to GST can be obtainedby cleaving the fusion protein with thrombin.

[0139] Examples of suitable inducible non-fusion E. coli expressionvectors are, inter alia, pTrc (Amann et al. (1988) Gene 69:301-315) andpET 11d (Studier et al., Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Targetgene expression of the pTrc vector is based on the transcription by hostRNA polymerase from a hybrid trp-lac fusion promoter. Target geneexpression from the pET 11d vector is based on transcription from aT7-gn10-lac fusion promoter which is mediated by a coexpressed viral RNApolymerase (T7 gn1). This viral polymerase is provided by the hoststrains BL21 (DE3) or HMS174 (DE3) by a resident λ prophage whichharbors a T7 gn1 gene under the transcriptional control of the lacUV 5promoter.

[0140] Other vectors which are suitable for use in prokaryotic organismsare known to the skilled worker; these vectors are, for example, in E.coli pLG338, pACYC184, the pBR series such as pBR322, the pUC seriessuch as pUC18 or pUC19, the M113mp series, pKC30, pRep4, pHS1, pHS2,pPLc236, pMBL24, pLG200, pUR290, pIN-III¹¹³-B1, λgt11 or pBdCI, inStreptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194or pBD214, in Corynebacterium pSA77 or pAJ667. A strategy of maximizingthe expression of recombinant protein is to express the protein in ahost bacterium whose ability to cleave the recombinant proteinproteolytically is disrupted (Gottesman, S., Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)119-128). A further strategy is to modify the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector, so that theindividual codons for each amino acid are those which are preferentiallyused in a bacterium selected for expression, such as C. glutamicum, etal. (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Modificationof these nucleic acid sequences according to the invention is carriedout by standard techniques of DNA synthesis.

[0141] In a further embodiment, the desaturase expression vector is ayeast expression vector. Examples of vectors for expression in the yeastS. cerevisiae include pYeDesaturasec1 (Baldari et al. (1987) Embo J.6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88(Schultz et al. (1987) Gene 54:113-123) and pYES2 (InvitrogenCorporation, San Diego, Calif.). Vectors and methods for theconstruction of vectors which are suitable for use in other fungi, suchas the filamentous fungi, include those which are described in detailin: van den Hondel, C. A. M. J. J., & Punt, P. J. (1991) “Gene transfersystems and vector development for filamentous fungi”, in: AppliedMolecular Genetics of fungi, J. F. Peberdy et al., Ed., pp. 1-28,Cambridge University Press: Cambridge, or in: More Gene Manipulations inFungi [J. W. Bennet & L. L. Lasure, Ed., pp. 396-428: Academic Press:San Diego]. Further suitable yeast vectors are, for example, pAG-1,YEp6, YEp13 or pEMBLYe23.

[0142] As an alternative, the desaturases according to the invention canbe expressed in insect cells using baculovirus expression vectors.Baculovirus vectors which are available for expressing proteins incultured insect cells (for example Sf9 cells) include the pAc series(Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series(Lucklow and Summers (1989) Virology 170:31-39).

[0143] The abovementioned vectors are just a short review of possiblesuitable vectors. Further plasmids are known to the skilled worker andare described, for example, in: Cloning Vectors (Ed. Pouwels, P. H., etal., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

[0144] In yet a further embodiment, a nucleic acid according to theinvention is expressed in mammalian cells using a mammalian expressionvector. Mammals for the purposes of the invention are to be understoodas all non-human mammals. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman etal. (1987) EMBO J. 6:187-195). When used in mammalian cells, the controlfunctions of the expression vector are frequently provided by viralregulatory elements. Promoters which are usually used are derived, forexample, from polyoma, adenovirus2, cytomegalovirus and Simian Virus 40.Other suitable expression systems for prokaryotic and eukaryotic cellscan be found in Chapters 16 and 17 of Sambrook, J., Fritsch, E. F., andManiatis, T., Molecular Cloning: A Laboratory Manual, 2nd Edition, ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

[0145] In another embodiment, the recombinant maamalian expressionvector can control the expression of the nucleic acid preferably in aspecific cell type (for example, tissue-specific regulatory elements areused for expressing the nucleic acid). Tissue-specific regulatoryelements are known in the art. Nonlimiting examples of suitabletissue-specific promoters are, inter alia, the albumen promoter(liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T-cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (for example neurofilament promoter; Byrne andRuddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund etal., (1985) Science 230:912-916) and mamma-specific promoters (forexample milk serum promoter; U.S. Pat. No. 4,873,316 and European PatentApplication document No. 264,166). Also included aredevelopment-regulated promoters, for example the mouse hox promoters(Kessel and Gruss (1990) Science 249:374-379) and the fetoproteinpromoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[0146] In a further embodiment, the desaturases according to theinvention can be expressed in single-celled plant cells (such as algae),see Falciatore et al., 1999, Marine Biotechnology 1 (3):239-251 andreferences cited therein, and plant cells from higher plants (forexample spermatophytes such as crops). Examples of plant expressionvectors include those which are described in detail in: Becker, D.,Kemper, E., Schell, J., and Masterson, R. (1992) “New plant binaryvectors with selectable markers located proximal to the left border”,Plant Mol. Biol. 20:1195-1197; and Bevan, M. W. (1984) “BinaryAgrobacterium vectors for plant transformation”, Nucl. Acids Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu,Academic Press, 1993, pp. 15-38.

[0147] A plant expression cassette preferably comprises regulatorysequences which can control gene expression in plant cells and which arelinked functionally so that each sequence can fulfil its function, suchas transcriptional termination, for example polyadenylation signals.Preferred polyadenylation signals are those derived from Agrobacteriumtumefaciens T-DNA, such as gene 3 of the Ti plasmid pTiACH5, which isknown as octopine synthase (Gielen et al., EMBO J. 3 (1984) 835 et seq.)or functional equivalents thereof, but all other terminators which arefunctionally active in plants are also suitable.

[0148] Since plant gene expression is very frequently not limited to thetranscription level, a plant expression cassette preferably comprisesother functionally linked sequences, such as translation enhancers, forexample the overdrive sequence, which contains the 5′-untranslatedtobacco mosaic virus leader sequence, which increases the protein/RNAratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-8711).

[0149] Plant gene expression must be linked functionally to a suitablepromoter which effects gene expression in a cell- or tissue-specificmanner with the correct timing. Preferred promoters are those which leadto constitutive expression (Benfey et al., EMBO J. 8 (1989) 2195-2202),such as those which are derived from plant viruses such as 35S CAMV(Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also U.S. Pat.No. 5,352,605 and WO 84/02913) or plant promoters such as the Rubiscosmall subunit promoter described in U.S. Pat. No. 4,962,028.

[0150] Other sequences which are preferred for use for functionallinkage in plant gene expression cassettes are targeting sequences whichare required for targeting the gene product into its correspoinding cellcompartment (for a review, see Kermode, Crit. Rev. Plant Sci. 15, 4(1996) 285-423 and references cited therein), for example into thevacuole, the nucleus, all types of plastids such as amyloplasts,chloroplasts, chromoplasts, the extracellular space, the mitochondria,the endoplasmic reticulum, elaioplasts, peroxisomes and othercompartments of plant cells.

[0151] Plant gene expression can also be facilitated via a chemicallyinducible promoter (for a review, see Gatz 1997, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 48:89-108). Chemically inducible promotersare particularly suitable when it is desired for gene expression to takeplace in a specific manner with regard to timing. Examples of suchpromoters are a salicylic acid-inducible promoter (WO 95/19443), atetracyclin-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404)and an ethanol-inducible promoter.

[0152] Other suitable promoters are promoters which respond to biotic orabiotic stress conditions, for example the pathogen-induced PRP1 genepromoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), theheat-inducible tomato hsp80 promoter (U.S. Pat. No. 5,187,267), the lowtemperature-inducible potato alpha-amylase promoter (WO 96/12814) or thewound-inducible pinII promoter (EP-A-0 375 091).

[0153] Promoters which are particularly preferred are those which leadto gene expression in tissues and organs in which lipid and oilbiosynthesis take place, in seed cells such as endosperm cells and cellsof the developing embryo. Promoters which are suitable are the oilseedrape napin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USPpromoter (Baeumlein et al., Mol Gen Genet, 1991, 225 (3):459-67), theArabidopsis oleosin promoter (WO 98/45461), the Phaseolus vulgarisphaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter(WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al., 1992,Plant Journal, 2 (2):233-9), and promoters which lead to theseed-specific expression in monocots such as maize, barley, wheat, rye,rice and the like. Notable promoters which are suitable are the barleylpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230) or thepromoters described in WO 99/16890 (promoters from the barley hordeingene, the rice glutelin gene, the rice oryzin gene, the rice prolamingene, the wheat gliadin gene, the wheat glutelin gene, the maize zeingene, the oat glutelin gene, the sorghum kasirin gene, the rye secalingene).

[0154] The multiparallel expression of desaturases according to theinvention, alone or in combination with other desaturases or elongases,may be desired in particular. The introduction of such expressioncassettes can be effected by a simultaneous transformation of aplurality of individual expression constructs or by combining aplurality of expression cassettes on one construct. Also, a plurality ofvectors can be transformed with in each case a plurality of expressioncassettes, and transferred to the host cell.

[0155] Promoters which are also particularly suitable are those whichlead to plastid-specific expression, since plastids are the compartmentin which the precursors and some end products of lipid biosynthesis aresynthesized. Suitable promoters such as the viral RNA polymerasepromoter are described in WO 95/16783 and WO 97/06250, and theArabidopsis clpP promoter, described in WO 99/46394.

[0156] The invention furthermore provides a recombinant expressionvector encompassing a DNA molecule according to the invention which iscloned into the expression vector in antisense orientation, i.e. the DNAmolecule is linked functionally to a regulatory sequence in such a waythat it allows the expression (by transcribing the DNA molecule) of anRNA molecule which is “antisense” to the desaturase mRNA. Regulatorysequences may be selected which are linked functionally to a nucleicacid cloned in antisense orientation and which control the continuousexpression of the antisense RNA molecule in a multiplicity of celltypes, for example, viral promoters and/or enhancers or regulatorysequences may be selected which control the constitutive,tissue-specific or cell-type-specific expression of antisense RNA. Theantisense expression vector may be present in the form of a recombinantplasmid, phagemid or attenuated virus in which the antisense nucleicacids are produced under the control of a highly effective regulatoryregion whose activity can be determined by the cell type into which thevector has been introduced. For an explanation of the regulation of geneexpression by means of antisense genes, see Weintraub, H., et al.,Antisense-RNA as a molecular tool for genetic analysis, Reviews—Trendsin Genetics, Vol. 1(1) 1986.

[0157] A further aspect of the invention relates to host cells intowhich a recombinant expression vector according to the invention hasbeen introduced. The terms “host cell” and “recombinant host cell” areused interchangeably in the present context. Naturally, these terms donot only refer to the particular target cell, but also to the progeny orpotential progeny of this cell. Since specific modifications may occurin subsequent generations owing to mutation or environmental effects,this progeny is not necessarily identical with the parental cell, butremains within the scope of the term as used in the present context.

[0158] The terms recombinant or transgene, for example recombinantexpression vector or recombinant host or host cells is to be understoodas meaning, for the purpose of the invention, that the nucleic acidsaccording to the invention and/or their natural regulatory sequences atthe 5′ and 3′ positions of the nucleic acids are not in their naturalenvironment, that is to say either the location of the sequences in theoriginal organism was altered or the nucleic acid sequences and/or theregulatory sequences were mutated in it or the nucleic acid sequencesaccording to the invention were transferred into an organism other thanthe original organism or their regulatory sequences. Combinations ofthese modifications are also possible. Natural environment is to beunderstood as meaning the location of a nucleic acid sequence in anorganism as it occurs in nature.

[0159] A host cell may be a prokaryotic or eukaryotic cell. For examplea desaturase can be expressed in bacterial cells such as C. glutamicum,insect cells, fungal cells or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells), algae, ciliates, plant cells, fungi orother microorganisms such as C. glutamicum. Other suitable host cellsare known to the skilled worker.

[0160] Vector DNA can be introduced into prokaryotic or eukaryotic cellsby conventional transformation or transfection techniques. The terms“transformation” and “transfection”, conjugation and transduction asused in the present context are intended to encompass a multiplicity ofmethods known in the art for introducing foreign nucleic acid (forexample DNA) into a host cell, including calcium phosphate or calciumchloride coprecipitation, DEAE-dextran-mediated transfection,lipofection, natural competence, chemically mediated transfer,electroporation or particle bombardment. Suitable methods for thetransformation or transfection of host cells, including plant cells, canbe found in Sambrook et al. (Molecular Cloning: A Laboratory Manual.,2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) and other laboratory text books,such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacteriumprotocols, Ed.: Gartland and Davey, Humana Press, Totowa, N.J.

[0161] It is known about the stable transfection of mammalian cells thatonly a small number of the cells integrate the foreign DNA into theirgenome, depending on the expression vector used and the transfectiontechnique used. To identify and select these integrants, a gene whichencodes a selectable marker (for example resistance to antibiotics) isusually introduced into the host cells together with the gene ofinterest. Preferred selectable markers encompass those which impartresistance to drugs such as G418, hygromycin and methotrexate, or, inplants, those which impart resistance to a herbicide such as glyphosateor glufosinate. Further suitable markers are, for example, markers whichencode genes which are involved in the biosynthetic pathways of, forexample, sugars or amino acids, such as β-galactosidase, ura3 or ilv2.Markers which encode genes such as luciferase, gfp or other fluorescencegenes are also suitable. These markers can be used in mutants in whichthese genes are not functional since they have been deleted for exampleby means of conventional methods. Furthermore, markers which encode anucleic acid which encodes a selectable marker can be introduced into ahost cell on the same vector as the one which encodes a desaturase, orcan be introduced on a separate vector. Cells which have beentransfected stably with the nucleic acid introduced can be identifiedfor example by drug selection (for example, cells which have theselectable marker integrated survive, whereas the other cells die).

[0162] To generate a microorganism with homologous recombination, avector is generated which contains at least one segment of a desaturasegene into which a deletion, addition or substitution has been introducedin order to modify the desaturase gene hereby, for example tofunctionally disrupt it. This desaturase gene is preferably aPhaeodactylum tricornutum desaturase gene, but a homolog or analog fromother organisms, even from mammalian, fungal or insect cells, can alsobe used. In a preferred embodiment, the vector is designed in such a waythat the endogenous desaturase gene is functionally disrupted (i.e. nolonger encodes a functional protein, also termed knock-out vector) uponhomologous recombination. As an alternative, the vector can be designedin such a way that the endogenous desaturase gene is mutated or modifiedotherwise upon homologous recombination while still encoding afunctional protein (for example, the upstream regulatory region can bemodified in such a way that this leads to a modification of theexpression of the endogenous desaturase). To generate a point mutationvia homologous recombination, DNA-RNA hybrids, which are also known aschimeraplasty, and which are known from Cole-Strauss et al., 1999,Nucleic Acids Research 27(5):1323-1330 and Kmiec, Gene therapy, 1999,American Scientist, 87(3):240-247 can also be used.

[0163] In the vector for homologous recombination, the modified segmentof the desaturase gene is flanked at its 5′ and 3′ end by additionalnucleic acid of the desaturase gene, so that homologous recombination ispossible between the exogenous desaturase gene which is present on thevector and an endogenous desaturase gene in a microorganism or plant.The additional flanking desaturase nucleic acid is sufficiently long forsuccessful homologous recombination with the endogenous gene. Usually,several hundred base pairs up to kilobases of flanking DNA (both on the5′ and on the 3′ end) are present in the vector (for a description ofvectors for homologous recombination, see, for example, Thomas, K. R.,and Capecchi, M. R. (1987) Cell 51:503 or for the recombination inPhyscomitrella patens on cDNA basis, see Strepp et al., 1998, Proc.Natl. Acad. Sci. USA 95 (8):4368-4373). The vector is introduced into amicroorganism or plant cell (for example by means of polyethyleneglycol-mediated DNA), and cells in which the desaturase gene introducedhas undergone homologous recombination with the endogenous desaturasegene are selected using techniques known in the art.

[0164] In another embodiment, recombinant organisms such asmicroorganisms can be generated which contain selected systems whichallow regulated expression of the gene introduced. The inclusion of adesaturase gene in a vector, where it is placed under the control of thelac operon, allows, for example, expression of the desaturase gene onlyin the presence of IPTG. These regulatory systems are known in the art.

[0165] A host cell according to the invention, such as a prokaryotic oreukaryotic host cell, growing either in culture or in a field, can beused for producing (i.e. expressing) a desaturase. In plants, analternative method can additionally be used by directly transferring DNAinto developing flowers via electroporation or Agrobacterium-mediatedgene transfer.

[0166] Accordingly, the invention furthermore provides methods ofproducing desaturases using the host cells according to the invention.In one embodiment, the method encompasses growing the host cellaccording to the invention (into which a recombinant expression vectorencoding a desaturase has been introduced or into whose genome a geneencoding a wild-type or modified desaturase has been introduced) in asuitable medium until the desaturase has been produced. In a furtherembodiment, the method encompasses isolating the desaturases from themedium or the host cell.

[0167] Host cells which are suitable in principle for taking up thenucleic acid according to the invention, the gene product according tothe invention or the vector according to the invention are allprokaryotic or eukaryotic organisms. The host organisms which are usedadvantageously are organisms such as bacteria, fungi, yeasts, animalcells or plant cells. Further advantageous organisms are animals or,preferably, plants or parts thereof. Fungi, yeasts or plants arepreferably used, especially preferably fungi or plants, very especiallypreferably plants such as oil crop plants which contain large amounts oflipid compounds, such as oilseed rape, evening primrose, canola, peanut,linseed, soybean, safflower, sunflower, borage, or plants such as maize,wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper,tagetes, Solanaceae plants such as potato, tobacco, egg-plant andtomato, Vicia species, pea, alfalfa, bush plants (coffee, cacao, tea),Salix species, trees (oil palm, coconut) and perennial grasses andfodder crops. Especially preferred plants according to the invention areoil crop plants such as soybean, peanut, oilseed rape, canola, linseed,evening primrose, sunflower, safflower, trees (oil palm, coconut).

[0168] D. Isolated Desaturase

[0169] A further aspect of the invention relates to isolated desaturasesand biologically active parts thereof. An “isolated” or “purified”protein or a biologically active part thereof is essentially free ofcellular material when it is produced by recombinant DNA techniques, orfree from chemical precursors or other chemicals when it is synthesizedchemically. The term “essentially free of cellular material” encompassesdesaturase preparations in which the protein is separated from cellularcomponents of the cells in which it is produced naturally orrecombinantly. In one embodiment, the term “essentially free of cellularmaterial” encompasses desaturase preparations with less thanapproximately 30% (based on the dry weight) of non-desaturase (alsoreferred to herein as “contaminating protein”), more preferably lessthan approximately 20% of non-desaturase, even more preferably less thanapproximately 10% of non-desaturase and most preferably less thanapproximately 5% of non-desaturase. If the desaturase or a biologicallyactive part thereof has been produced recombinantly, it is alsoessentially free of culture medium, i.e. the culture medium amounts toless than approximately 20%, more preferably less than approximately 10%and most preferably less than approximately 5% of the volume of theprotein preparation. The term “essentially free from chemical precursorsor other chemicals” encompasses desaturase preparations in which theprotein is separate from chemical precursors or other chemicals whichare involved in the synthesis of the protein. In one embodiment, theterm “essentially free of chemical precursors or other chemicals”encompasses desaturase preparations with less than approximately 30%(based on the dry weight) of chemical precursors or non-desaturasechemicals, more preferably less than approximately 20% of chemicalprecursors or non-desaturase chemicals, even more preferably less thanapproximately 10% of chemical precursors or non-desaturase chemicals andmost preferably less than approximately 5% of chemical precursors ornon-desaturase chemicals. In preferred embodiments, isolated proteins orbiologically active parts thereof exhibit no contaminating proteins fromthe same organisms from which the desaturase originates. These proteinsare usually produced by recombinant expression, for example,Phaeodactylum tricornutum desaturase in plants such as Physcomitrellapatens or abovementioned microorganisms, for example bacteria such as E.coli, Bacillus subtilis, C. glutamicum, fungi such as Mortierella,yeasts such as Saccharomyces, or ciliates such as Colpidium or algaesuch as Phaeodactylum.

[0170] An isolated desaturase according to the invention or a partthereof can also participate in the metabolism of compounds required forthe synthesis of cell membranes in Phaeodactylum tricornutum or in thetransport of molecules via these membranes. In preferred embodiments,the protein or the part thereof encompasses an amino acid sequence whichhas sufficient homology with an amino acid sequence of SEQ ID NO: 2, 4,6 or 12 for the protein or part thereof to retain the ability toparticipate in the metabolism of compounds required for the synthesis ofcell membranes in Phaeodactylum tricornutum or in the transport ofmolecules via these membranes. The part of the protein is preferably abiologically active part as described herein. In a further preferredembodiment, a desaturase according to the invention has one of the aminoacid sequences shown in SEQ ID NO: 2, 4, 6 or 12. In a further preferredembodiment, the desaturase has an amino acid sequence which is encodedby a nucleotide sequence which hybridizes with a nucleotide sequence ofSEQ ID NO: 1, 3, 5 or 11, for example under stringent conditions. In yetanother preferred embodiment, the desaturase has an amino acid sequencewhich is encoded by a nucleotide sequence which has at leastapproximately 50 to 60%, preferably at least approximately 60 to 70%,more preferably at least approximately 70 to 80%, 80 to 90%, 90 to 95%,and even more preferably at least approximately 96%, 97%, 98%, 99% ormore homology with one of the amino acid sequences of SEQ ID NO: 2, 4, 6or 18. The desaturase preferred according to the invention preferablyalso has at least one of the desaturase activities described herein. Forexample, a desaturase preferred according to the invention encompassesan amino acid sequence encoded by a nucleotide sequence which hybridizeswith a nucleotide sequence of SEQ ID NO: 1, 3, 5 or 11, for exampleunder stringent conditions, and which can participate in the metabolismof compounds required for the synthesis of cell membranes inPhaeodactylum tricornutum or in the transport of molecules via thesemembranes and is capable of introducing a double bond into a fatty acidwith one, two, three or four double bonds and a chain length of C₁₈, C₂₀or C₂₂.

[0171] In other embodiments, the desaturase is essentially homologouswith an amino acid sequence of SEQ ID NO: 2, 4 or 6 and retains thefunctional activity of the protein of one of the sequences of SEQ ID NO:2, 4 or 6, the amino acid sequence differing, however, owing to naturalvariation or mutagenesis as described in detail in the above subsectionI. In a further embodiment, the desaturase is, accordingly, a proteinencompassing an amino acid sequence which has at least approximately 50to 60% homology, preferably approximately 60 to 70% homology and morepreferably at least approximately 70 to 80%, 80 to 90%, 90 to 95%homology and most preferably at least approximately 96%, 97%, 98%, 99%or more homology with a complete amino acid sequence of SEQ ID NO: 2, 4or 6 and has at least one of the desaturase activities described herein.In another embodiment, the invention relates to a complete Phaeodactylumtricornutum protein which is essentially homologous with a completeamino acid sequence of SEQ ID NO: 2, 4 or 6.

[0172] Biologically active parts of a desaturase encompass peptidesencompassing amino acid sequences derived from the amino acid sequenceof a desaturase, for example an amino acid sequence shown in SEQ ID NO:2, 4 or 6 or the amino acid sequence of a protein which is homologouswith a desaturase, which peptides have fewer amino acids than thefull-length desaturase or the full-length protein which is homologouswith a desaturase and have at least one activity of a desaturase.Biologically active parts (peptides, for example peptides with a lengthof, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 ormore amino acids) usually encompass a domain or a motif with at leastone activity of a desaturase. Moreover, other biologically active partsin which other regions of the protein are deleted can be generated byrecombinant techniques and examined for one or more of the activitiesdescribed herein. The biologically active parts of the desaturasepreferably encompass one or more selected domains/motifs or partsthereof with biological activity.

[0173] Desaturases are preferably produced by recombinant DNAtechniques. For example, a nucleic acid molecule encoding the protein iscloned into an expression vector (as described above), the expressionvector is introduced into a host cell (as described above), and thedesaturase is expressed in the host cell. The desaturase can then beisolated from the cells by a suitable purification scheme using standardtechniques of protein purification. As an alternative to the recombinantexpression, a desaturase, a desaturase polypeptide or a desaturasepeptide can be synthesized chemically by means of standard techniques ofpeptide synthesis. Moreover, native desaturase can be isolated fromcells (for example endotheliol cells), for example using ananti-desaturase antibody which can be raised by standard techniques,using a desaturase according to the invention or a fragment thereof.

[0174] The invention also provides chimeric desaturase proteins ordesaturase fusion proteins. As used in the present context, a “chimericdesaturase proteins” or “desaturase fusion protein” encompasses adesaturase polypeptide which is bound functionally to a non-desaturasepolypeptide. A “desaturase polypeptide” refers to a polypeptide with anamino acid sequence which corresponds to a desaturase, whereas a“non-desaturase polypeptide” refers to a polypeptide with an amino acidsequence which corresponds to a protein which is essentially nothomologous with the desaturase, for example a protein which differs fromthe desaturase and which originates from the same or another organism.Within the fusion protein, the term “linked functionally” is understoodas meaning that the desaturase polypeptide and the non-desaturasepolypeptide are fused to each other in such a way that both sequencesfulfil the predicted function which has been ascribed to the sequenceused. The non-desaturase polypeptide can be fused to the N terminus orthe C terminus of the desaturase polypeptide. In one embodiment, thefusion protein is, for example, an EST-desaturase fusion protein inwhich the desaturase sequences are fused to the C terminus of the GSTsequences. These fusion proteins can facilitate the purification of therecombinant desaturases. In a further embodiment, the fusion protein isa desaturase which has a heterologous signal sequence (N terminus). Inspecific host cells (for example mammalian host cells), the expressionand/or secretion of a desaturase can be increased by using aheterologous signal sequence.

[0175] A chimeric desaturase protein or desaturase fusion proteinaccording to the invention is produced by standard recombinant DNAtechniques. For example, DNA fragments which encode differentpolypeptide sequences are ligated to each other in-frame usingconventional techniques, for example by employing blunt ends oroverhanging ends for ligation, restriction enzyme cleavage for providingsuitable ends, filling up cohesive ends, as required, treatment withalkaline phosphatase to avoid undesired linkages, and enzymationligation. In a further embodiment, the fusion gene can be synthesized byconventional techniques including DNA synthesizers. As an alternative,PCR amplification of gene fragments can be carried out using anchorprimers which generate complementary overhangs between successive genefragments which can subsequently be hybridized with each other andreamplified to give rise to a chimeric gene sequence (see, for example,Current Protocols in Molecular Biology, Ed. Ausubel et al., John Wiley &Sons: 1992). Moreover, a large number of expression vectors whichalready encode a fusion unit (for example a GST polypeptide) arecommercially available. A desaturase-encoding nucleic acid can be clonedinto such an expression vector so that the fusion unit is linkedin-frame to the desaturase protein.

[0176] Desaturase homologs can be generated by mutagenesis, for exampleby specific point mutation or by truncating the desaturase. The term“homologs” as used in the present context refers to a variant form ofthe desaturase which acts as agonist or antagonist with the desaturaseactivity. A desaturase agonist can essentially retain the same activityas the desaturase, or some of the biological activities of thedesaturase. A desaturase antagonist can inhibit one or more activitiesof the naturally occurring desaturase form, for example by competitivebinding to an upstream or downstream element of the metabolic cascadefor cell membrane components which encompass the desaturase, or bybinding to a desaturase which mediates the transport of compounds viacell membranes, thus inhibiting translocation. In an alternativeembodiment, desaturase homologs can be identified by screeningcombinatory libraries of desaturase mutants, for example truncatedmutants, with regard to desaturase agonist or antagonist activity. Inone embodiment, a variegated library of desaturase variants is generatedat nucleic acid level by combinatory mutagenesis and encoded by avariegated genetic library. A variegated library of desaturase variantscan be generated for example by enzymatic ligation of a mixture ofsynthetic oligonucleotides into gene sequences so that a degenerate setof potential desaturase sequences can be expressed as individualpolypeptides or, alternatively, as a set of larger fusion proteins (forexample for phage display) which comprise this set of desaturasesequences. There is a multiplicity of methods which can be used forgenerating libraries of potential desaturase homologs from a degenerateoligonucleotide sequence. The chemical synthesis of a degenerate genesequence can be carried out in a DNA synthesizer, and the synthetic genecan then be ligated into a suitable expression vector. The use of thedegenerate set of genes allows all sequences which encode the desiredset of potential desaturase sequences to be provided in a mixture.Methods for the synthesis of degenerate oligonucleotides are known inthe art (see, for example, Narang, S. A. (1983) Tetrahedron 39:3;Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984)Science 198:1056; Ike et al. (1983) Nucleic Acids Res. 11:477).

[0177] In addition, libraries of desaturase fragments can be used forgenerating a variegated population of desaturase fragments for screeningand for the subsequent selection of homologs of a desaturase. In oneembodiment, a library of fragments of the coding sequence can begenerated by treating a double-strand PCR fragment of a codingdesaturase sequence with a nuclease under conditions under whichdouble-strand breaks only occur approximately once per molecule,denaturing the double-stranded DNA, renaturing the DNA with theformation of double-stranded DNA which can encompass sense/antisensepairs of various products with double-strand breaks, removal ofsingle-stranded sections from newly formed duplices by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. Using this method, an expression library can be derived whichencodes N-terminal, C-terminal and internal desaturase fragments ofvarious sizes.

[0178] A number of techniques for screening gene products in combinatorylibraries which have been generated by point mutation or truncation andfor screening cDNA libraries for gene products with a selected propertyare known in the art. These techniques can be adapted to rapid screeningof the gene libraries which have been generated by combinatorymutagenesis of desaturase homologs. The most frequently used techniquesfor screening large gene libraries which can be subjected tohigh-throughput analysis usually encompass cloning the gene library intoreplicable expression vectors, transforming suitable cells with theresulting vector library, and expressing the combinatory genes underconditions under which detecting the desired activity facilitates theisolation of the vector encoding the gene whose product has beendetected. Recursive ensemble mutagenesis (REM), a novel technique whichincreases the frequency of functional mutants in the libraries, can beused in combination with the screening assays for identifying desaturasehomologs (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

[0179] A further known technique for modifying catalytic properties ofenzymes or the genes encoding them is gene shuffling (see, for example,Stemmer, PNAS 1994, 91: 10747-10751, WO 97/20078 or WO 98/13487), whichis a combination of gene fragments where this new combination canadditionally be varied by erroneous polymerase chain reactions thuscreating a high sequence diversity to be assayed. However, theprerequisite for using such an approach is a suitable screening systemfor testing the resulting gene diversity for functionality.

[0180] A screening method which identifies a PUFA-dependent enzymeactivity or activities, is a prerequisite in particular for screeningdesaturase activities. As regards desaturase activities with aspecificity for PUFAs, the toxicity of arachidonic acid in the presenceof a toxic metabolyte (here: salicylic acid or salicylic acidderivatives) can be exploited in Mucor species which can be transformedwith desired gene constructs by known transformation methods (Eroshin etal., Mikrobiologiya, Vol. 65, No.1 1996, pages 31-36), to carry out agrowth-based primary screening. Resulting clones can then be analyzedfor their lipid constituents by means of gas chromatography and massspectroscopy in order to identify the nature and quantity of startingmaterials and products.

[0181] In a further embodiment, cell-based assays can be made use of foranalyzing a variegated desaturase library using further processes knownin the art.

[0182] E. Uses and Processes/Methods According to the Invention

[0183] The nucleic acid molecules, proteins, protein homologs, fusionproteins, primers, vectors and host cells described herein can be usedin one or more of the processes/methods which follow: identification ofPhaeodactylum and related organisms, genome mapping of organisms whichare related to Phaeodactylum tricornutum, identification andlocalization of Phaeodactylum tricornutum sequences of interest,evolutionary studies, determination of desaturase protein regionsrequired for the function, modulation of a desaturase activity,modulation of the metabolism of one or more cell membrane components,modulation of the transmembrane transport of one or more compounds, andmodulation of the cellular production of a desired compound such as afine chemical. The desaturase nucleic acid molecules according to theinvention have a multiplicity of uses. Firstly, they can be used foridentifying an organism as Phaeodactylum tricornutum or a close relativethereof. They can also be used for identifying the presence ofPhaeodactylum tricornutum or of a relative thereof in a mixed populationof microorganisms. The, invention provides the nucleic acid sequences ofa series of Phaeodactylum tricornutum genes; the presence or absence ofthis organism can be determined by screening the extracted genomic DNAof a culture of a uniform or mixed population of microorganisms understringent conditions with a probe covering a region of a Phaeodactylumtricornutum gene or parts thereof, which gene is unique to thisorganism. Phaeodactylum tricornutum itself is used for the commercialproduction of polyunsaturated acids and is additionally suitable for theproduction of PUFAs, also in other organisms, in particular when it isintended for the resulting PUFAs also to be incorporated into thetriacylglycerol fraction.

[0184] Furthermore, the nucleic acid and protein molecules according tothe invention can act as markers for specific regions of the genome.This is suitable not only for mapping the genome, but also forfunctional Phaeodactylum tricornutum proteins. To identify the genomeregion to which a certain DNA-binding protein of Phaeodactylumtricornutum binds, it might be possible, for example, to fragment thePhaeodactylum tricornutum genome, and the fragments could be incubatedwith the DNA-binding protein. Those which bind the protein canadditionally be screened with the nucleic acid molecules according tothe invention, preferably with readily detectable markers; the bindingof such a nucleic acid molecule to the genome fragment makes possiblethe localization of the fragment on the genome map of Phaeodactylumtricornutum and, if this is carried out repeatedly with differentenzymes, facilitates a rapid determination of the nucleic acid sequenceto which the protein binds. Moreover, the nucleic acid moleculesaccording to the invention can have sufficient homology with thesequences of related species for these nucleic acid molecules to be ableto act as markers for the construction of a genomic map in related fungior algae.

[0185] The desaturase nucleic acid molecules according to the inventionare also suitable for evolutionary studies and studies of the proteinstructure. The metabolic and transport processes in which the moleculesaccording to the invention are involved are utilized by many prokaryoticand eukaryotic cells; the evolutionary degree of relatedness of theorganisms can be determined by comparing the sequences of the nucleicacid molecules according to the invention with those which encodesimilar enzymes from other organisms. Accordingly, such a comparisonallows the determination of which sequence regions are conserved andwhich are not conserved, and this may be helpful when determiningregions of the protein which are essential for enzyme function. Thistype of determination is valuable for protein engineering studies andmay provide a clue of how much mutagenesis the protein can toleratewithout losing its function.

[0186] Manipulation of the desaturase nucleic acid molecules accordingto the invention can lead to the production of desaturases withfunctional differences to the wild-type desaturases. The efficiency oractivity of these proteins can be improved, they can be present in thecell in larger numbers than usual, or their efficiency or activity canbe reduced. An improved efficiency or activity means, for example, thatthe enzyme has a higher selectivity and/or activity, preferably anactivity which is at least 10% higher, especially preferably an activitywhich is at least 20% higher, very especially preferably an activitywhich is at least 30% higher than that of the original enzyme.

[0187] There exists a series of mechanisms by which modification of adesaturase according to the invention can directly affect the yield,production and/or production efficiency of a fine chemical comprisingsuch a modified protein. Obtaining fine chemical compounds from culturesof ciliates, algae or fungi on a large scale is significantly improvedwhen the cell secretes the desired compounds, since these compounds canbe isolated readily from the culture medium (in contrast to extractionfrom the biomass of the cultured cells). Otherwise, purification can beimproved when the cell stores compounds in-vivo in a specializedcompartment with a sort of concentration mechanism. In plants whichexpress desaturases, an increased transport may lead to betterdistribution within the plant tissue and the plant organs. Increasingthe number or the activity of transporter molecules which export finechemicals from the cell may allow the quantity of the fine chemicalsproduced, which is present in the extracellular medium, to be increased,thus facilitating harvesting and purification or, in the case of plants,more efficient distribution. In contrast, increased amounts ofcofactors, precursor molecules and intermediates for the suitablebiosynthetic pathways are required for efficient overproduction of oneor more fine chemicals. Increasing the number and/or the activity oftransporter proteins involved in the import of nutrients such as carbonsources (i.e. sugars), nitrogen sources (i.e. amino acids, ammoniumsalts), phosphate and sulfur can improve the production of a finechemical owing to the elimination of all limitations of the nutrientsavailable in the biosynthetic process. Fatty acids such as PUFAs andlipids comprising PUFAs are desirable fine chemicals themselves;optimizing the activity or increasing the number of one or moredesaturases according to the invention involved in the biosynthesis ofthese compounds, or disrupting the activity of one or more desaturasesinvolved in the catabolism of these compounds, can thus increase theyield, production and/or production efficiency of fatty acids and lipidmolecules in ciliates, algae, plants, fungi, yeasts or othermicroorganisms.

[0188] The manipulation of one or more desaturase genes according to theinvention can likewise lead to desaturases with modified activitieswhich indirectly affect the production of one or more desired finechemicals from algae, plants, ciliates or fungi. The normal biochemicalmetabolic processes leaked, for example, to the production of amultiplicity of waste products (for example hydrogen peroxide and otherreactive oxygen species) which can actively disrupt these metabolicprocesses (for example, peroxynitrite is known to nitrate tyrosine sidechains, thus inactivating some enzymes with tyrosin in the active center(Groves, J. T. (1999) Curr. Opin. Chem. Biol. 3(2);226-235)). Whilethese waste products are normally excreted, the cells used forfermentative production on a large scale are optimized for theoverproduction of one or more fine chemicals and can therefore producemore waste products than is customary for a wild-type cell. Optimizingthe activity of one or more desaturases according to the inventioninvolved in the export of waste molecules allows the improvement of theviability of the cell and the maintenance of an efficient metabolicactivity. Also, the presence of high intracellular amounts of thedesired fine chemical can in fact be toxic to the cell, so that theviability of the cell can be improved by increasing the ability of thecell to secrete these compounds.

[0189] Furthermore, the desaturases according to the invention can bemanipulated in such a way that the relative amounts of various lipidsand fatty acid molecules are modified. This can have a decisive effecton the lipid composition of the cell membrane. Since each lipid type hasdifferent physical properties, a modification of the lipid compositionof the membrane can significantly modify membrane fluidity. Changes inmembrane fluidity can affect the transport of molecules via the membranewhich, as explained above, can modify the export of waste products or ofthe fine chemical produced or the import of nutrients which arerequired. These changes in membrane fluidity can also have a decisiveeffect on cell integrity; cells with comparatively weaker membranes aremore susceptible to abiotic and biotic stress conditions which candamage or kill the cell. Manipulation of desaturases involved in theproduction of fatty acids and lipids for membrane synthesis so that theresulting membrane has a membrane composition which is more susceptibleto the environmental conditions prevailing in the cultures used for theproduction of fine chemicals should allow more cells to survive andmultiply. Larger numbers of producing cells should manifest themselvesin greater yields, higher production or higher production efficiency ofthe fine chemical from the culture.

[0190] The abovementioned mutagenesis strategies for desaturasesintended to lead to elevated yields of a fine chemical are not to beconstrued as limiting; variations of these strategies are readilyobvious to the skilled worker. Using these mechanisms, and with the aidof the mechanisms disclosed herein, the nucleic acid and proteinmolecules according to the invention can be used for generating algae,ciliates, plants, animals, fungi or other microorganisms such as C.glutamicum, which express mutated desaturase nucleic acid and proteinmolecules so that the yield, production and/or production efficiency ofa desired compound is improved. This desired compound can be any naturalproduct of algae, ciliates, plants, animals, fungi or bacteria whichencompasses the end products of biosynthetic pathways and intermediatesof naturally occurring metabolic pathways, and also molecules which donot naturally occur in the metabolism of these cells, but which areproduced by the cells according to the invention.

[0191] A further embodiment according to the invention is a process forthe production of PUFAs, which comprises culturing an organism whichcontains a nucleic acid according to the invention, a gene constructaccording to the invention or a vector according to the invention whichencode a polypeptide which elongates C₁₈-, C₂₀- or C₂₂-fatty acids withat least two double bonds in the fatty acid molecule by at least twocarbon atoms under conditions under which PUFAs are produced in theorganism. PUFAs produced by this process can be isolated by harvestingthe organisms either from the culture in which they grow or from thefield, and disrupting and/or extracting the harvested material with anorganic solvent. The oil, which contains lipids, phospholipids,sphingolipids, glycolipids, triacylglycerols and/or free fatty acidswith a higher PUFA content can be isolated from this solvent. The freefatty acids with a higher PUFA content can be isolated by basic or acidhydrolysis of the lipids, phospholipids, sphingolipids, glycolipids andtriacylglycerols. A higher PUFA content means at least 5%, preferably10%, especially preferably 20%, very especially preferably 40% morePUFAs than the original organism which does not have additional nucleicacid encoding the desaturase according to the invention.

[0192] The PUFAs produced by this process are preferably C₁₈- orC₂₀₋₂₂-fatty acid molecules with at least two double bonds in the fattyacid molecule, preferably three, four, in combination with a furtherelongase and a Δ4-desaturase five or six double bonds. These C₁₈- orC₂₀₋₂₂-fatty acid molecules can be isolated from the organism in theform of an oil, lipid or a free fatty acid. Examples of suitableorganisms are those mentioned above. Preferred organisms are transgenicplants.

[0193] An embodiment according to the invention are oils, lipids orfatty acids or fractions thereof which have been prepared by theabove-described process, especially preferably an oil, a lipid or afatty acid composition comprising PUFAs and originating from transgenicplants.

[0194] A further embodiment according to the invention is the use of theoil, lipid or fatty acid composition in feeds, foods, cosmetics orpharmaceuticals.

[0195] The invention further relates to a method of identifying anantaganist or agonist of desaturases, comprising

[0196] a) contacting the cells which express the polypeptide of thepresent invention with a candidate substance;

[0197] b) testing the desaturate activity;

[0198] c) comparing the desaturase activity with a standard activity inthe absence of the candidate material, where an increase in thedesaturase activity beyond the standard indicates that the candidatematerial is an agonist and a reduction in the desaturase activityindicates that the candidate material is an antagonist.

[0199] The candidate substance mentioned can be a substance which hasbeen synthesized chemically or produced by microbes and can occur, forexample, in cell extracts of, for example, plants, animals ormicroorganisms. Moreover, the substance mentioned, while being known inthe prior art, may not be known as yet as increasing or reversing theactivity of the desaturases. The reaction mixture can be a cell-freeextract or encompass a cell or cell culture. Suitable methods are knownto the skilled worker and are described in general terms for example inAlberts, Molecular Biology the cell, 3rd Edition (1994), for exampleChapter 17. The substances mentioned can be added for example to thereaction mixture or the culture medium or else injected into the cellsor sprayed onto a plant.

[0200] When a sample comprising an active substance by the methodaccording to the invention has been identified, it is either possibledirectly to isolate the substance from the original sample or else thesample can be divided into various groups, for example when they consistof a multiplicity of various components, in order to reduce the numberof the various substances per sample and then to repeat the methodaccording to the invention with such a “subset” of the original sample.Depending on the complexity of the sample, the above-described steps canbe repeated repeatedly, preferably until the sample identified inaccordance with the method according to the invention only stillcontains a small number of substances, or only one substance.Preferably, the substance identified in accordance with the methodaccording to the invention, or derivatives of the substance, areformulated further so that it is suitable for use in plant breeding orin plant cell or tissue culture.

[0201] The substances which have been assayed and identified inaccordance with the method according to the invention can be: expressionlibraries, for example cDNA expression libraries, peptides, proteins,nucleic acids, antibodies, small organic substances, hormones, PNAs orthe like (Milner, Nature Medicin 1 (1995), 879-880; Hupp, Cell. 83(1995), 237-245; Gibbs, Cell. 79 (1994), 193-198 and references citedtherein). These substances can also be functional derivatives or analogsof the known inhibors or activators. Methods of preparing chemicalderivatives or analogs are known to the skilled worker. The derivativesand analogs mentioned can be assayed in accordance with prior-artmethods. Moreover, computer-aided design or peptidomimetics can be usedfor producing suitable derivatives and analogs. The cell or the tissuewhich can be used for the method according to the invention ispreferably a host cell according to the invention, a plant cellaccording to the invention or a plant tissue as described in theabovementioned embodiments.

[0202] Accordingly, the present invention also relates to a substancewhich has been identified in accordance with the above methods accordingto the invention. The substance is, for example, a homolog of thedesaturases according to the invention. Homologs of the desaturases canbe generated by mutagenesis, for example by point mutation or deletionof the desaturases. The term “homolog” as used in the present contextdenotes a variant form of the desaturases which acts as agonist orantagonist for the activity of the desaturases. An agonist can haveessentially the same or part of the biological activity of thedesaturases. An antagonist of the desaturases can inhibit one or moreactivities of the naturally occurring forms of the desaturases, forexample can undergo competitive banding to a downstream or upstreammember of the fatty acid synthesis metabolic pathways, which include thedesaturases, or can bind to desaturases and thus reduce or inhibit theactivity.

[0203] Moreover, the present invention also relates to an antibody or afragment thereof as are described herein, which antibody or fragmentinhibits the activity of the desaturases according to the invention.

[0204] In one aspect, the present invention relates to an antibody whichspecifically recognizes, or binds to, the above-described agonist orantagonist according to the invention.

[0205] A further aspect relates to a composition comprising theantibody, the stop identified by the method according to the inventionor the antisense molecule.

[0206] In a further embodiment, the present invention relates to a kitcomprising the nucleic acid according to the invention, the geneconstruct according to the invention, the amino acid sequence accordingto the invention, the antisense nucleic acid molecule according to theinvention, the antibody and/or composition according to the invention,an antagonist or agonist prepared by the method according to theinvention, and/or oils, lipids and/or fatty acids according to theinvention or a fraction thereof. Equally, the kit can comprise the hostcells, organisms, plants according to the invention or parts thereof,harvestable parts of the plants according to the invention orpropagation material or else the antagonist or agonist according to theinvention. The components of the kit of the present invention can bepackaged in suitable containers, for example with or in buffers or othersolutions. One or more of the abovementioned components may be packagedin one and the same container. In addition, or as an alternative, one ormore of the abovementioned components can be adsorbed onto a solidsurface, for example nitrocellulose filters, glass sheets, chips, nylonmembranes or microtiter plates. The kit can be used for any of themethods and embodiments described herein, for example for the productionof host cells, transgenic plants, for the detection of homologoussequences, for the identification of antagonists or agonists and thelike. Furthermore, the kit can comprise instructions for the use of thekit for one of the abovementioned applications.

[0207] The present invention is illustrated in greater detail by theexamples which follow, and which must not be construed as limiting. Thecontent of any references, patent applications, patents and publishedpatent applications cited in the present patent application is herewithincorporated by reference.

EXAMPLES SECTION Example 1 General Methods

[0208] a) General Cloning Methods:

[0209] Cloning methods such as, for example, restriction cleavages,agarose gel electrophoresis, purification of DNA fragments, transfer ofnucleic acids to nitrocellulose and nylon membranes, linkage of DNAfragments, transformation of Escherichia coli and yeast cells, theculture of bacteria and the sequence analysis of recombinant DNA werecarried out as described in Sambrook et al. (1989) (Cold Spring HarborLaboratory Press: ISBN 0-87969-309-6) or Kaiser, Michaelis and Mitchell(1994) “Methods in Yeast Genetics” (Cold Spring Harbor Laboratory Press:ISBN 0-87969-451-3). The transformation and culture of algae such asChlorella or Phaeodactylum are carried out as described by El-Sheekh(1999), Biologia Plantarum 42:209-216; Apt et al. (1996) Molecular andGeneral Genetics 252 (5):872-9.

[0210] b) Chemicals

[0211] Unless otherwise specified in the text, the chemicals used wereobtained in analytical quality from Fluka (Neu-Ulm), Merck (Darmstadt),Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Solutionswere supplied using pure pyrogen-free water, referred to in thefollowing text as H₂O, on a Milli-Q water system water purification unit(Millipore, Eschborn). Restriction endonucleases, DNA-modifying enzymesand molecular biology kits were obtained from AGS (Heidelberg), Amersham(Braunschweig), Biometra (Göttingen), Boehringer (Mannheim), Genomed(Bad Oeynhausen), New England Biolabs (Schwalbach/Taunus), Novagen(Madison, Wis., USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg),Qiagen (Hilden) and Stratagene (Amsterdam, the Netherlands). Unlessotherwise specified, they were used following the manufacturer'sinstructions.

[0212] c) Cell Material

[0213] The isolated nucleic acid sequences according to the inventionare present in the genome of a Phaeodactylum tricornutum UTEX646 strain,which is available from the algae collection of the University of Texas,Austin.

[0214]Phaeodactylum tricornutum was cultured at 25° C. at a light/darkphoto period of 14:10 hours at 22° C. and 35 microEinstein (correspondsto micromol of photons per square meter and second) in glass tubes intowhich air was passed in from the bottom.

[0215] The culture medium used for Phaeodactylum tricornutum was the f/2culture medium supplemented with 10% organic medium of Guillard, R. R.L. (1975; Culture of phytoplankton for feeding marine invertebrates. In:Smith, W. L. and Chanley, M. H. (Eds.) Culture of marine Invertebrateanimals, NY Plenum Press, pp. 29-60.): It comprises

[0216] 995.5 ml of (artificial) sea water 1 ml of NaNO₃ (75 g/l), 1 mlof NaH₂PO₄ (5 g/l), 1 ml of trace element solution, 1 ml of Tris/Cl pH8.0, 0.5 ml of f/2 vitamin solution

[0217] Trace element solution: Na₂EDTA (4.36 g/l), FeCl₃ (3.15 g/l),Primary trace elements: CuSO₄ (10 g/l), ZnSO₄ (22 g/l), CoCl₂ (10 g/l),MnCl₂ (18 g/l), NaMoO₄ (6.3 g/l) f/2 vitamin solution: biotin: 10 mg/l,thiamine 200 mg/l, vitamin B12 0.1 mg/l org medium: sodium acetate (1g/l), glucose (6 g/l), sodium succinate (3 g/l), Bacto-tryptone (4 g/l),yeast extract (2 g/l)

Example 2 Isolation of Total DNA from Phaeodactylum tricornutum UTEX646for Hybridization Experiments

[0218] The details of the isolation of total DNA refer to the work-up ofplant material with a fresh weight of one gram.

[0219] CTAB buffer: 2% (w/v) N-acetyl-N,N,N-trimethylammonium bromide(CTAB); 100 mM Tris-HCl, pH 8.0; 1.4 M NaCl; 20 mM EDTA.

[0220] N-Laurylsarcosine buffer: 10% (w/v) of N-laurylsarcosine; 100 mMTris-HCl, pH 8.0; 20 mM EDTA.

[0221]Phaeodactylum tricornutum cell material was triturated in a mortarunder liquid nitrogen to give a fine powder which was transferred into 2ml Eppendorf vessels. The frozen plant material was then covered with alayer of 1 ml of break buffer (1 ml of CTAB buffer, 100 ml ofN-laurylsarcosine buffer, 20 ml of β-mercaptoethanol and 10 ml ofproteinase K solution, 10 mg/ml) and incubated at 60° C. for one hourwith continuous shaking. The homogenate obtained was distributed intotwo Eppendorf vessels (2 ml) and extracted twice by shaking with anequal volume of chloroform/isoamyl alcohol (24:1). For phase separation,centrifugation was carried out at 8000×g and RT (=room temperature=˜23°C.) for 15 minutes in each case. The DNA was then precipitated for 30minutes at −70° C. using ice-cold isopropanol. The precipitated DNA wassedimented for 30 minutes at 10 000 g at 4° C. and resuspended in 180 mlof TE buffer (Sambrook et al., 1989, Cold Spring Harbor LaboratoryPress: ISBN 0-87969-309-6). For further purification, the DNA wastreated with NaCl (final concentration 1.2 M) and reprecipitated for 30minutes at −70° C. using twice the volume of absolute ethanol. After awash step with 70% strength ethanol, the DNA was dried and subsequentlytaken up in 50 ml of H₂O+RNase (final concentration 50 mg/ml). The DNAwas dissolved overnight at 4° C., and the RNase cleavage wassubsequently carried out for 1 hour at 37° C. The DNA was stored at 4°C.

Example 3 Isolation of Total RNA and Poly(A)⁺ RNA from Plants andPhaeodactylum tricornutum

[0222] Total RNA was isolated from plants such as linseed and oilseedrape and the like by a method described by Logemann et al. (1987, Anal.Biochem. 163, 21). The total RNA from moss can be obtained fromprotonema tissue using the GTC method (Reski et al., 1994, Mol. Gen.Genet., 244:352-359).

[0223] RNA Isolation of Phaeodactylum tricornutum:

[0224] Frozen samples of algae (−70° C.) were triturated in an ice-coldmortar under liquid nitrogen to give a fine powder. 2 volumes ofhomogenization medium (12.024 g of sorbitol, 40.0 ml of 1M Tris-HCl, pH9 (0.2 M); 12.0 ml of 5 M NaCl (0.3 M), 8.0 ml of 250 mM EDTA, 761.0 mgof EGTA, 40.0 ml of 10% SDS were made up to 200 ml with H₂O and the pHwas brought to 8.5) and 4 volumes of phenol with 0.2% mercaptoethanolwere added to the frozen cell powder at 40 to 50° C. while mixingthoroughly. Then, 2 volumes of chloroform were added and the mixture wasstirred vigorously for 15 minutes. The mixture was centrifuged for 10minutes at 10 000 g and the aqueous phase was extracted withphenol/chloroform (2 volumes) and subsequently extracted withchloroform.

[0225] The resulting volume of the aqueous phase was treated with{fraction (1/20)} volume of 4 M sodium acetate (pH 6) and 1 volume ofisopropanol (ice-cold), and the nucleic acids were precipitatedovernight at −20° C. The mixture was then centrifuged for 30 minutes at10 000 g and the supernatant pipetted off. This was followed by a washstep with 70% strength EtOH and another centrifugation. The sediment wastaken up in Tris-borate buffer (80 mM Tris-borate buffer, 10 mM EDTA, pH7.0). The supernatant was then treated with ⅓ volume of 8 M LiCl, mixedand incubated for 30 minutes at 4° C. After recentrifugation, thesediment was washed with 70% strength ethanol, centrifuged and thesediment was dissolved in RNase-free water.

[0226] Poly(A)⁺ RNA was isolated using Dyna Beads® (Dynal, Oslo, Norway)following the instructions in the manufacturer's protocol.

[0227] After the RNA or poly(A)⁺ RNA concentration had been determined,the RNA was precipitated by adding {fraction (1/10)} volume of 3 Msodium acetate, pH 4.6, and 2 volumes of ethanol and stored at −70° C.

[0228] For the analysis, 20 μg portions of RNA were separated in aformaldehyde-containing 1.5% strength agarose gel and transferred tonylon membranes (Hybond, Amersham). Specific transcripts were detectedas described by Amasino ((1986) Anal. Biochem. 152, 304)).

Example 4 Construction of the cDNA Library

[0229] To construct the cDNA library from Phaeodactylum tricornutum, thefirst-strand synthesis was carried out using murine leukaemia virusreverse transcriptase (Roche, Mannheim, Germany) and oligo-d(T) primers,while the second-strand synthesis was carried out by incubation with DNApolymerase I, Klenow enzyme and RNase H cleavage at 12° C. (2 hours),16° C. (1 hour) and 22° C. (1 hour). The reaction was quenched byincubation at 65° C. (10 minutes) and subsequently transferred to ice.Double-stranded DNA molecules were made blunt-ended with T4 DNApolymerase (Roche, Mannheim) at 37° C. (30 minutes). The nucleotideswere removed by extraction with phenol/chloroform and Sephadex G50 spincolumns. EcoRI/XhoI adapters (Pharmacia, Freiburg, Germany) were ligatedto the cDNA ends by means of T4 DNA ligase (Roche, 12° C., overnight),recut with XhoI and phosphorylated by incubation with polynucleotidekinase (Roche, 37° C., 30 min). This mixture was subjected to separationon a low-melting agarose gel. DNA molecules with over 300 base pairswere eluted from the gel, extracted with phenol, concentrated on ElutipD columns (Schleicher and Schüll, Dassel, Germany) and ligated to vectorarms and packaged into lambda-ZAP-Express phages using the Gigapack Goldkit (Stratagene, Amsterdam, the Netherlands), using the manufacturer'smaterial and following their instructions.

Example 5 DNA Sequencing and Computer Analysis

[0230] cDNA libraries as described in Example 4 were used for DNAsequencing by standard methods, in particular the chain terminationmethod using the ABI PRISM Big Dye Terminator Cycle Sequencing ReadyReaction Kit (Perkin-Elmer, Weiterstadt, Germany). Sequencing of randomclones which had been singled out was carried out following preparativeplasmid preparation from cDNA libraries via in-vivo mass excision andretransformation of DH10B on agar plates (details on materials andprotocol: Stratagene, Amsterdam, the Netherlands). Plasmid DNA wasprepared from E. coli cultures grown overnight in Luria brothsupplemented with ampicillin (see Sambrook et al. (1989) (Cold SpringHarbor Laboratory Press: ISBN 0-87969-309-6)) using a Qiagen DNApreparation robot (Qiagen, Hilden) following the manufacturer'sprotocols. Sequencing primers with the following nucleotide sequenceswere used: 5′-CAGGAAACAGCTATGACC-3′ 5′-CTAAAGGGAACAAAAGCTG-3′5′-TGTAAAACGACGGCCAGT-3′

[0231] The sequences were processed and annotated using the EST-MAXstandard software package, which is commercially available from Bio-Max(Munich, Germany). Exploiting comparative algorithms, and using thesearch sequence shown in SEQ ID NO: 8, homologous genes were searchedfor using the BLAST program (Altschul et al. (1997) “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402). Two sequences from Phaeodactylumtricornutum with homologies with the Physcomitrella patens searchsequence were characterized in greater detail.

Example 5a Isolation of Phaeodactylum tricornutum Desaturases ViaPolymerase Chain Reaction with the Aid of Degenerate Oligonucleotides

[0232] Published desaturases allow motifs to be identified which aretypical of Δ5- and Δ6-desaturases. Oligonucleotide sequences withpossible variations are shown in the following text. Underneath theoligonucleotide sequence, the amino acid from which the base combinationcan be derived is shown in the one-letter code. For example, A/G meansthat either an A or a G is randomly incorporated at this position in theoligonucleotide when the unit is synthesized, since the base tripletderived from the corresponding amino acid can either be AAA or AAG. TheDNA sequence may also contain an inosine (i) if the determination of abase at this position permits three or four different bases owing to thegenetic code. The following sequences and primers can be used:5′-forward primer: F1a: TGG TGG AA A/G TGG  AAi     CA T/C AA F1b: TGGTGG AA A/G TGG  ACi     CA T/C AA F1a: W   W   K      W   N/T       H   K/N F1b: W   W   K      W    K        H    K/N F2a:  Gi TGG AA A/G GAi  A/C AiCA T/C  AA F2b:  Gi TGG AA A/G TTG  A/C Ai CA T/C  AA F2a:G/W  W   K     E/D  K/Q/N   H      K/N F2b:G/W  W   K      W   K/Q/N   H      K/N F3a: T A/T i    TTG  AAi  A/C AA/G C/A G/A i  CA F3b: T A/T i    TTG  AAi  A/C A A/G CAi        CA F3a:W           W   K/N  H/N       R/Q        H F3b:Y           W   K/N  H/N       R/Q        H F4a:       GTi TGG A A/TG/A  GA A/G    CA A/G CA F4b:       GTi TGG A A/T G/A  A/T A T/C CA A/GCA F4a:       V    W    K/M      E          Q     H F4b:      V    W    K/M      N/Y        Q     H F5a1:       CA T/C TA T/CTGG AA A/G AA T/C CA G C F5a1:       CA T/C TA T/C TGG AA A/G AA T/C CAA C F5a1:        H      Y      W   K      N      Q   H/Q F6a:TTG   TTG  AAi  A/C A A/G  AA i       CA T/C AA F6a:W      W   K/N  H/N   K/N         H   K/N 3′-reverse primer primer R1b:       GG  A/G AA  iAG  G/A TG  G/A TG   T/C TC R1b:        GG  A/GAA  iAA  G/A TG  G/A TG   T/C TC R1a:       P       F    L        H      H        E R1b:       P       F    F        H      H        E R2a1:       AA    iAG  A/G TG  A/G TG   iA C/T   iA/G  T/C TG R2a2:        AAT/C AA  A/G TG  A/G TG   iA C/T   iA/G  T/C TG R2a1:       F      L       H       H     V/I       V/A     Q R3a1:       AT  iTG    iGG A/G AA  iAA    A/G TG  A/G TG R3a2:        AT  A/GTT iGG A/G AA  iAA    A/G TG  A/G TG R3a3:        AT  iTG    iGG A/GAA  iAG    A/G TG  A/G TG R3a4:        AT  A/G TT iGG A/G AA  iAG    A/GTG  A/G TG R3a1:        I/M H/Q     P   F       F         H       HR3a2:        I/M  N      P   F       L         H       H R4a1:            CT    iGG  A/G AA  iA A/G   A/G TG  A/G TG R4a2:            GA    iGG  A/G AA  iA A/G   A/G TG  A/G TG R4a3:            GT    iGG  A/G AA  iA A/G   A/G TG  A/G TG R4a1:       =   T/R/S   P     F      F/L        H       H R5a1:       AA iAA     A/G TG  A/G TG  T/C TC    T/A/G AT T/C TG R5a2:       AA iAG     A/G TG  A/G TG  T/C TC    T/A/G AT T/C TG R5a1:        F F            H       H       E     I           Q R5a2:        F L            H       H       E     I           Q R6a1:        T     iGG iA  A/G   iAA   A/G TG A/G TG  iAC R6a1:        T     iGG iA  A/G   iAG   A/G TG A/G TG  iAC R6a1:       T/N    P    L         F/L     H       H    V

[0233] Owing to various possibilities of variations, a large number ofderived oligonucleotides are possible, but surprisingly it has beenfound that oligonucleotides shown can be particularly suitable forisolating desaturases.

[0234] The primers can be employed for polymerase chain reactions in allcombinations. Individual combinations allowed desaturase fragments to beisolated when the following conditions were taken into consideration:for PCR reactions, in each case 10 nMol of primer and 10 ng of a plasmidlibrary obtained by in-vivo excision were employed. It was possible toisolate the plasmid library from the phage library following theprotocols of the manufacturer (Stratagene). The PCR reaction was carriedout in a thermocycler (Biometra) using Pfu DNA polymerase (Stratagene)and the following temperature program: 3 minutes at 96° C. followed by35 cycles with 30 seconds at 96° C., 30 seconds at 55° C. and 1 minuteat 72° C. After the first step at 55° C., the annealing temperature waslowered stepwise by in each case 3° C., and, after the fifth cycle, anannealing temperature of 40° C. was retained. Finally, a 10-minute-cycleat 72° C. was carried out, and the reaction was stopped by cooling to 4°C.

[0235] The primer combinations F6a and R4a2 are shown underlined in thetext, and it was possible to exploit them successfully for isolating adesaturase fragment. It was possible to verify the resulting fragment bysequencing; it showed homologies with Streptomyces coelicolor desaturasewith the Genbank Accession No. T36617. The homology was obtained withthe aid of the BLASTP program. The alignment is shown in FIG. 4.Identities of 34% and a homology of 43% with sequence T36617 wererevealed. The DNA fragment was employed in accordance with the inventionas shown in Example 7 in a hybridization experiment for isolating afull-length gene under standard conditions.

[0236] The coding region of a DNA sequence isolated in this way wasobtained by translating the genetic code into a polypeptide sequence.SEQ ID NO: 3 shows a sequence 1434 base pairs in length which wasisolated by the method described. The sequence has a start codon inpositions 1 to 3 and a stop codon in positions 1432-1434 and it waspossible to translate it into a polypeptide 477 amino acids in length.In the alignment with the gene sequence described in WO 98/46763, it wasfound that a nonidentical, but homologous, Phaeodactylum tricornutumfragment encoding 87 amino acids had previously been described. However,WO 98/46763 discloses neither a complete, functionally active desaturasenor position or substrate specificity. This is also made clear by thefact that homologies with both the Δ5- and Δ6-desaturase fromMortierella alpina are reported without indicating a specific function.The sequence according to the invention, in contrast, encodes afunctionally active Δ6-acyl lipid desaturase.

Example 6 Identification of DNA Sequences Encoding Phaeodactylumtricornutum Desaturases

[0237] The full-length sequence of the Δ6-acyl lipid desaturase Pp_des6AJ222980 (NCBI Genbank Accession No.) from the moss Physcomitrellapatens (see also Table 1) and the Δ12-acyl lipid desaturase sequence(Table 1, see Ma_des12) from Mortierella alpina AF110509 (AF110509 NCBIGenbank Accession No.) were employed for sequence alignment with the aidof the TBLASTN search algorithm.

[0238] The EST sequences PT0010070010R, PT001072031R and PT001078032Rwere first considered as target gene among further candidate genes owingto weak homologies with the search sequences from Physcomitrella andMortierella. FIGS. 1, 2 and 2 a show the result of the two EST sequencesfound. The sequences found are part of the nucleic acids according tothe invention of SEQ ID NO: 1 (gene name: Pt_des5, inventors' owndatabase No. PT001078032R), SEQ ID NO: 5. (gene name: Pt_des12,inventors' own database No. PT0010070010R) and SEQ ID NO: 11 (gene name:Pt_des12.2, inventor's own database No. PT001072031R). Letters indicateidentical amino acids, while the plus symbol indicates a chemicallysimilar amino acid. The identities and homologies of all sequences foundin accordance with the invention can be seen from the summary in Table2.

[0239] Desaturases can have cytochrome b5 domains which also occur inother genes which do not code for desaturases. Thus, cytochrome b5domains show high homologies, even though the gene functions aredifferent. Within weakly conserved regions, desaturases can only beidentified as putative candidate genes and must be tested for the enzymeactivity and position specificity of the enzymatic function. Forexample, various hydroxylases, acetylenases and epoxygenases, likedesaturases, also show histidine box motifs, so that a specific functionmust be proven experimentally and only the additional verification ofthe double bond makes possible a guaranteed enzyme activity and positionspecificity of a desaturase. Surprisingly, it has been found that Δ6-and Δ5-desaturases according to the invention have particularly suitablesubstrate specificities and are particularly suitable for beingexploited, in combination with a Physcomitrella Δ6-elongase, forproducing polyunsaturated fatty acids such as arachidonic acid,eicosapentaenoic acid and docosahexanoic acid.

[0240] Sequencing of the full cDNA fragment from clone PT001078032Rrevealed a sequence 1652 base pairs in length. The sequence encodes apolypeptide of 469 amino acids shown in SEQ ID NO: 2. It was obtained bytranslating the genetic code of SEQ ID NO: 1 with a start codon in basepair position 115-117 and with a stop codon in base pair position1522-1524. The clone comprises a complete desaturase polypeptide, as canbe seen from the sequence alignment in FIG. 3. Lines denote identicalamino acids, while colons and dots represent chemically exchangeable,i.e. chemically equivalent, amino acids. The alignment was carried outusing Henikoff & Henikoff's BLOSUM62 substitution matrix ((1992) Aminoacid substitution matrices from protein blocks. Proc. Natl. Acad. Sci.USA 89: 10915-10919). Parameters used: Gap Weight: 8; Average Match:2.912, Length Weight: 2, Average Mismatch: −2.003. FIG. 6 and FIG. 7show the alignment of the MA_des12 peptide sequence with the sequencesfound.

[0241] Sequencing of the complete cDNA fragment from clone PT0010070010Rrevealed a sequence 1651 base pairs in length and shown in SEQ ID NO: 5with a start codon in position 67-69 and a stop codon in position1552-1554. The polypeptide sequence according to the invention is shownin SEQ ID NO: 6.

[0242] Sequencing the complete cDNA fragment identified from clonePT0010072031R revealed a sequence 1526 base pairs in length and shown inSEQ ID NO: 11 with a start codon in position 92-94 and a stop codon inposition 1400-1402. The polypeptide sequence according to the inventionis shown in SEQ ID NO: 12.

[0243] Table 2 shows the identities and homologies of desaturasesaccording to the invention with each other and with the Physcomitrellapatens and Mortierella alpina desaturase. The data were obtained withthe aid of the Bestfit program under given parameters as definedhereinbelow as a subprogram of the following software: Wisconsin PackageVersion 10.0 (Genetics Computer Group (GCG), Madison, Wis., USA).Henikoff, S. and Henikoff, J. G. (1992). Amino acid substitutionmatrices from protein blocks. Proc. Natl. Acad. Sci. USA 89:10915-10919.

[0244] Furthermore, FIG. 5 shows the alignment of the Physcomitrellapatens Δ6-acyl lipid desaturase with the polypeptide sequence of clonePt_des6. TABLE 2 Homology/ Search sequence Search sequence identity in %Pp_des6 Ma_des12 Pt_des5 34.92/26.37 n.d. Pt_des6 50.69/41.06 n.d.Pt_des12 n.d. 48.58/38.92 Pt_des12.2 n.d. 48.37/41.60

[0245] With the aid of the algorithm TBLASTN 2.0.10: Altschul et al.1997, “Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs”, Nucleic Acids Res. 25:3389-3402, sequences with thehighest sequence homology or identity were identified via a localdatabase alignment. The results are shown in Table 2A hereinbelow. TABLE2A Homologs with the highest sequence homologies or identities withpolypeptide sequences according to the invention of SEQ ID NO. 2, 4, 6or 12 Search se- Search Search Search quence Homology/ sequence sequencesequence PT_(—) identity (%) PT001070010R PT001072031R PT001078032R des6L26296: 50%/37% n.d. n.d. n.d. Fad2 A. thaliana U86072 n.d. 51/40 n.d.n.d. Petroselinum crispum Fad2 AL358652 n.d. n.d. 45/30 n.d. L. majorputative desaturase AB020032 n.d. n.d. n.d. 53/38 M. alpina Δ6desaturase

Example 7 Identification of Genes by Means of Hybridization

[0246] Gene Sequences can be Used for Identifying Homologous orHeterologous Genes from cDNA or Genomic Libraries.

[0247] Homologous genes (i.e. full-length cDNA clones which arehomologous, or homologs) can be isolated via nucleic acid hybridizationusing, for example, cDNA libraries: the method can be used in particularfor isolating functionally active full-length genes of those shown inSEQ ID NO: 3. Depending on the frequency of the gene of interest, 100000 up to 1 000 000 recombinant bacteriophages are plated out andtransferred to a nylon membrane. After denaturation with alkali, the DNAwas immobilized on the membrane, for example by UV crosslinking.Hybridization is performed under high-stringency conditions. Thehybridization and the wash steps are carried out in aqueous solution atan ionic strength of 1 M NaCl and a temperature of 68° C. Hybridizationprobes were generated for example by labeling with radioactive (³²P-)nick transcription (High Prime, Roche, Mannheim, Germany). The signalsare detected by autoradiography.

[0248] Partially homologous or heterologous genes which are related butnot identical can be identified analogously to the process describedabove using low-stringency hybridization and wash conditions. For theaqueous hybridization, the ionic strength was usually kept at 1 M NaCl,and-the temperature was lowered gradually from 68 to 42° C.

[0249] The isolation of gene sequences which only exhibit homologieswith an individual domain of, for example, 10 to 20 amino acids can becarried out using synthetic, radiolabeled oligonucleotide probes.Radiolabeled oligonucleotides are generated by phosphorylating the 5′end of two complementary oligonucleotides with T4 polynucleotide kinase.The complementary oligonucleotides are hybridized and ligated to eachother to give rise to concatemers. The double-stranded concatemers areradiolabeled for example by nick transcription. Hybridization is usuallycarried out under low-stringency conditions using high oligonucleotideconcentrations.

[0250] Oligonucleotide Hybridization Solution:

[0251] 6×SSC

[0252] 0.01 M sodium phosphate

[0253] 1 mM EDTA (pH 8)

[0254] 0.5% SDS

[0255] 100 μg/ml denatured salmon sperm DNA

[0256] 0.1% dry low-fat milk

[0257] During the hybridization, the temperature is lowered stepwise to5 to 10° C. below the calculated oligonucleotide Tm or down to roomtemperature (unless otherwise specified, RT=˜23° C. in all experiments),followed by wash steps and autoradiography. Washing is carried out atextremely low stringency, for example three wash steps using 4×SSC.Further details are as described by Sambrook, J., et al. (1989),“Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor LaboratoryPress, or Ausubel, F. M., et al. (1994) “Current Protocols in MolecularBiology”, John Wiley & Sons.

Example 8 Identification of Target Genes by Screening ExpressionLibraries with Antibodies

[0258] To generate recombinant protein, for example E. coli, cDNAsequences were used (for example Qiagen QIAexpress pQE system). Therecombinant proteins were then affinity-purified, usually via Ni-NTAaffinity chromatography (Qiagen). The recombinant proteins were thenused for raising specific antibodies, for example using standardtechniques for immunizing rabbits. The antibodies were thenaffinity-purified using an Ni-NTA column which is desaturated withrecombinant antigen, as described by Gu et al., (1994) BioTechniques17:257-262. The antibody can then be used for screening expression cDNAlibraries by immunological screening (Sambrook, J., et al. (1989),“Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor LaboratoryPress, or Ausubel, F. M., et al. (1994) “Current Protocols in MolecularBiology”, John Wiley & Sons).

Example 9 Transformation of Agrobacterium

[0259] Agrobacterium-mediated plant transformation can be effected forexample using the Agrobacterium tumefaciens strain GV3101-(pMP90-)(Koncz and Schell, Mol. Gen. Genet. 204 (1986) 383-396) orLBA4404-(Clontech) or C58C1 pGV2260 (Deblaere et al 1984, Nucl. AcidsRes. 13, 4777-4788). The transformation can be carried out by standardtransformation techniques (Deblaere et al., 1984, IBID.).

Example 10 Plant Transformation

[0260] Agrobacterium-mediated plant transformation can be effected usingstandard transformation and regeneration techniques (Gelvin, Stanton B.,Schilperoort, Robert A., Plant Molecular Biology Manual, 2nd Ed.,Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuc ZentraleSignatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R., Thompson, JohnE., Methods in Plant Molecular Biology and Biotechnology, Boca Raton:CRC Press, 1993, 360 pp., ISBN 0-8493-5164-2).

[0261] For example, oilseed rape can be transformed by means ofcotyledon or hypocotyl transformation (Moloney et al., Plant Cell 8(1989) 238-242; De Block et al., Plant Physiol. 91 (1989) 694-701). Theuse of antibiotics for the selection of agrobacteria and plants dependson the binary vector and the agrobacterial strain used for thetransformation. The selection of oilseed rape is normally carried outusing kanamycin as selectable plant marker.

[0262] Agrobacterium-mediated gene transfer in linseed (Linumusitatissimum) can be carried out for example using a techniquedescribed by Mlynarova et al. (1994) Plant Cell Report 13:282-285.

[0263] The transformation of soybean can be carried out for exampleusing a technique described in EP-A-0 0424 047 (Pioneer Hi-BredInternational) or in EP-A-0 0397 687, U.S. Pat. No. 5,376,543, U.S. Pat.No. 5,169,770 (University Toledo).

[0264] Plant transformation using particle bombardment, polyethyleneglycol-mediated DNA uptake or via the silicon carbonate fiber techniqueis described, for example, by Freeling and Walbot “The maize handbook”(1993) ISBN 3-540-97826-7, Springer Verlag New York).

Example 11 Plasmids for Plant Transformation

[0265] Binary vectors such as pBinAR (Höfgen and Willmitzer, PlantScience 66 (1990) 221-230) or PGPTV (Becker et al 1992, Plant Mol. Biol.20:1195-1197) or derivatives of these can be used for transformingplants. The binary vectors can be constructed by ligating the cDNA insense or antisense orientation into T-DNA. 5′ of the cDNA, a plantpromoter activates cDNA transcription. A polyadenylation sequence islocated 3′ of the cDNA. The binary vectors can contain different markergenes. In particular, the nptII marker gene encoding kanamycinresistance mediated by neomycin phosphotransferase can be exchanged forthe herbicide-resistant form of an acetolactate synthase gene(abbreviation: AHAS or ALS). The ALS gene is described in Ott et al., J.Mol. Biol. 1996, 263:359-360. The v-ATPase-c1 promoter can be clonedinto plasmid pBin19 or pGPTV and exploited for marker gene expression bycloning it before the ALS coding region. The promoter stated correspondsto a 1153 base pair fragment from Beta vulgaris (Plant Mol. Biol., 1999,39:463-475). Both sulfonylureas and imidazolinones such as imazethapyror sulfonylureas can be used as antimetabolites for selection.

[0266] Tissue-specific expression can be achieved by using atissue-specific promoter. For example, seed-specific expression can beachieved by cloning the DC3 or the LeB4 or the USP promoter or thephaseolin promoter 5′ of the cDNA. Any other seed-specific promoterelement can also be used, such as, for example, the napin or arcelinpromoter (Goossens et al. 1999, Plant Phys. 120(4):1095-1103 andGerhardt et al. 2000, Biochimica et Biophysica Acta 1490(1-2):87-98).The CaMV 35S promoter or a v-ATPase C1 promoter can be used forconstitutive expression in imtact plants.

[0267] In particular, genes encoding desaturases and elongases can becloned into a binary vector one after the other by constructing severalexpression cassettes in order to imitate the metabolic pathway inplants.

[0268] Within an expression cassette, the protein to be expressed can betargeted into a cellular compartment using a signal peptide, for examplefor plastids, mitochondria or the endoplasmic reticulum (Kermode, Crit.Rev. Plant Sci. 15, 4 (1996) 285-423). The signal peptide is cloned 5′in-frame with the cDNA in order to achieve subcellular localization ofthe fusion protein.

[0269] Examples of multiexpression cassettes are given hereinbelow.

[0270] I.) Promoter-Terminator Cassettes

[0271] Expression cassettes are composed of at least two functionalunits, such as a promoter and a terminator. Further desired genesequences such as targeting sequences, coding regions of genes or partsthereof etc. can be inserted between promoter and terminator. In orderto construct expression cassettes, promoters and terminators (USPpromoter: Baeumlein et al., Mol. Gen. Genet., 1991, 225 (3):459-67); OCSterminator: Gielen et al. EMBO J. 3 (1984) 835 et seq.) are isolatedwith the aid of polymerase chain reaction and tailor-made as desiredwith flanking sequences based on synthetic oligonucleotides.

[0272] Examples of the oligonucleotides which can be used are thefollowing: USP1 front: CCGGAATTCGGCGCGCCGAGCTCCTCGAGCAAATTTACACATTGCCAUSP2 front: CCGGAATTCGGCGCGCCGAGCTCCTCGAGCAAATTTACACATTGCCA USP3 front:CCGGAATTCGGCGCGCCGAGCTCCTCGAGCAAATTTACACATTGCCA USP1 back:AAAACTGCAGGCGGCCGCCCACCGCGGTGGGCTGGCTATGAAGAAATT USP2 back:CGCGGATCCGCTGGCTATGAAGAAATT USP3 back:TCCCCCGGGATCGATGCCGGCAGATCTGCTGGCTATGAAGAAATT OCS1 front:AAAACTGCAGTCTAGAAGGCCTCCTGCTTTAATGAGATAT OCS2 front:CGCGGATCCGATATCGGGCCCGCTAGCGTTAACCCTGCTTTAATGAGATAT OCS3 front:TCCCCCGGGCCATGGCCTGCTTTAATGAGATAT OCS1 back:CCCAAGCTTGGCGCGCCGAGCTCGAATTCGTCGACGGACAATCAGTAAATTGA OCS2 back:CCCAAGCTTGGCGCGCCGAGCTCGAATTCGTCGACGGACAATCAGTAAATTGA OCS3 back:CCCAAGCTTGGCGCGCCGAGCTCGTCGACGGACAATCAGTAAATTGA

[0273] The methods are known to the skilled worker in the art and aregenerally known from the literature.

[0274] In a first step, a promoter and a terminator are amplified viaPCR. Then, the terminator is cloned into a recipient plasmid and, in asecond step, the promoter is inserted before the terminator. This givesan expression cassette on a carrier plasmid. Plasmids pUT1, pUT2 andpUT3 are generated on the basis of plasmid pUC19.

[0275] The constructs are defined in accordance with the invention inSEQ ID NO: 13, 14 and 15. Based on pUC19, they comprise the USP promoterand the OCS terminator. Based on these plasmids, construct pUT12 isgenerated by cutting pUT1 with SalI/ScaI and cutting pUT2 withXhoI/ScaI. The fragments in the expression cassette are ligated andtransformed into E. coli XLI blue MRF. After singling outampicillin-resistant colonies, DNA is prepared, and those clones whichcomprise two expression cassettes are identified by restrictionanalysis. The XhoI/SalI ligation of compatible ends has eliminated thetwo cleavage sites XhoI and SalI between the expression cassettes. Thisgives rise to plasmid pUT12, which is defined in SEQ ID NO: 16. pUT12 issubsequently cut again with SalI/ScaI and pUT3 with XhoI/ScaI. Thefragments comprising the expression cassettes are ligated andtransformed into E. coli XLI blue MRF. After singling outampicillin-resistant columns, DNA is prepared, and those clones whichcomprise three expression cassettes are identified by restrictionanalysis. In this manner, a set of multiexpression cassettes is createdwhich can be exploited for inserting the desired DNA and is described inTable 3 and can additionally incorporate further expression cassettes.

[0276] They comprise the following elements: TABLE 3 pUC19 Cleavagesites for the Multiple Cleavage sites behind the derivate USP promotercloning cleavage sites OCS terminator pUT1 EcoRI/Asc/SacI/XhoIBstXl/NotI/PstI/XbaI/StuI SalI/EcoRI/SacI/AscI/ HindIII pUT2EcoRI/AscI/SacI/XhoI BamHI/EcoRV/ApaI/NheI/HpaI SalI/EcoRI/SacI/AscI/HindIII pUT3 EcoRI/AscI/SacI/XhoI BglII/NaeI/ClaI/SmaI/NcoISalI/SacI/AscI/HindIII pUT12 EcoRI/AscI/SacI/XhoIBstXl/NotI/PstI/XbaI/StuI SalI/EcoRI/SacI/AscI/ Double and HindIIIexpression BamHI/EcoRV/ApaI/NheI/HpaI cassette pUT123EcoRI/AscI/SacI/XhoI 1.BstXI/NotI/PstI/XbaI/StuI SalI/SacI/AscI/HindIIITriple and expression 2.BamHI/EcoRV/ApaI/NheI cassette HpaI and3.BglII/NaeI/ClaI/SmaI/NcoI

[0277] Furthermore, further multiexpression cassettes can be generatedand employed for the seed-specific gene expression, as described and asspecified in greater detail in Table 4, with the aid of the

[0278] i) USP promoter or with the aid of the

[0279] ii) approx. 700 base pair 3′ fragment of the LeB4 promoter orwith the aid of the

[0280] iii) DC3 promoter.

[0281] The DC3 promoter is described in Thomas, Plant Cell 1996,263:359-368 and consists merely of the region −117 to +27, which is whyit therefore constitutes one of the smallest known seed-specificpromoters. The expression cassettes can comprise several copies of thesame promoter or else be constructed via three different promoters.TABLE 4 Multiple expression cassettes Plasmid name Cleavage sites beforethe pUC19 the respective Multiple Cleavage sites behind derivativepromoter cloning cleavage sites the OCS terminator pUT1EcoRI/AscI/SacI/XhoI (1) BstXI/NotI/PstI/XbaI/StuI SalI/EcoRI/SacI/AscI/(pUC19 with HindIII USP-OCS1) pDCT EcoRI/AscI/SacI/XhoI (2)BamHI/EcoRV/ApaI/NheI/ SalI/EcoRI/SacI/AscI/ (pUC19 with HpaI HindIIIDC3-OCS) pLeBT EcoRI/AscI/SacI/XhoI (3) BglII/NaeI/ClaI/SmaI/NcoISalI/SacI/AscI/HindIII (pUC19-with LeB4(700)-OCS) pUD12EcoRI/AscI/SacI/XhoI (1) BstXI/NotI/PstI/XbaI/StuI SalI/EcoRI/SacI/AscI/(pUC 19 with and HindIII USP-OCS1 and (2) BamHI/EcoRV/ApaI/NheI/ withDC3-OCS) HpaI pUDL123 EcoRI/AscI/SacI/XhoI (1) BstXI/NotI/PstI/XbaI/StuISalI/SacI/AscI/HindIII Triple expression and cassette (2)BamHI/(EcoRV*)/ApaI/ (pUC19 with NheI/HpaI and USP/DC3 and (3)BglII/NaeI/ClaI/SmaI/NcoI LeB4-700)

[0282] Further promoters for multi-gene constructs can be generatedanalogously, in particular using the

[0283] a) 2.7 kb fragment of the LeB4 promoter or with the aid of the

[0284] b) phaseolin promoter or with the aid of the

[0285] c) constitutive v-ATPase c1 promoter.

[0286] It may be particularly desirable to use further especiallysuitable promoters for constructing seed-specific multi-expressioncassettes such as, for example, the napin promoter or the arcelin-5promoter.

[0287] ii) Generation of Expression Construct in pUC19 Derivatives orpGPTV Derivatives Receiving Promoter and Terminator and Comprised inCombination With Desired Gene Sequences for PUFA Gene Expression inPlant Expression Cassettes.

[0288] Using AscI, multi-expression cassettes can be inserted directlyfrom pUC19 derivatives of Table 3 into the vector pGPTV+AscI (see iii.))via the AscI cleavage site and are available for inserting target genes.The gene constructs in question (pBUT1 is shown in SEQUENCE ID NO: 20,pBUT2 is shown in SEQUENCE ID NO: 21, pBUT3 is shown in SEQUENCE ID NO:22, pBUT 12 is shown in SEQUENCE ID NO: 22 and pBUT123 is shown inSEQUENCE ID NO: 24) are available in accordance with the invention inkit form. As an alternative, gene sequences can be inserted into thepUC19-based expression cassettes and inserted into pGPTV+AscI in theform of an AscI fragment.

[0289] In pUT12, the Δ6-elongase Pp_PSE1 is first inserted into thefirst cassette via BstXI and XbaI. Then, the moss Δ6-desaturase(Pp_des6) is inserted into the second cassette via BamHI/NaeI. Thisgives rise to the construct pUT-ED. The AscI fragment from plasmidpUT-ED is inserted into the AscI-cut vector pGPTV+AscI, and theorientation of the inserted fragment is determined by restriction orsequencing. This gives rise to plasmid pB-DHGLA, whose complete sequenceis shown in SEQUENCE ID NO. 25. The coding region of the PhyscomitrellaΔ6-elongase is shown in SEQUENCE ID NO. 26, that of the PhyscomitrellaΔ6-desaturase in SEQUENCE ID NO: 27.

[0290] In pUT123, the Δ6-elongase Pp_PSE1 is first inserted into thefirst cassette via BstXI and XbaI. Then, the moss Δ6-desaturase(Pp_des6) is inverted into the second cassette via BamHI/NaeI, and,finally, the Phaeodactylum Δ5-desaturase (Pt_des5) is inserted into thethird cassette via BglII. The triple construct is given the name pARA1.Taking into consideration sequence-specific restriction cleavage sites,further expression cassettes termed pARA2, pARA3 and pARA4 can begenerated, as shown in Table 5.

[0291] The AscI fragment from plasmid pARA1 is inserted into theAscI-cut vector pGPTV+AscI and the orientation of the inserted fragmentis determined by means of restriction or sequencing. The completesequence of the resulting plasmid pBARA1 is shown in SEQUENCE ID NO. 28.The coding region of the Physcomitrella Δ6-elongase is shown in SEQUENCEID NO. 29, that of the Physcomitrella Δ6-desaturase in SEQUENCE ID NO:30 and that of the Phaeodactylum tricornutum Δ5-desaturase in SEQUENCEID NO: 31. TABLE 5 Combinations of desaturases and elongases GenePlasmid Δ6-desaturase Δ5-desaturase Δ6-elongase 1 PUT-ED Pp_des6 —Pp_PSE1 2 pARA1 Pt_des6 Pt_des5 Pp_PSE1 3 pARA2 Pt_des6 Ce_des5 Pp_PSE14 pARA3 Pt_des6 Ce_des5 Pp_PSE1 5 pARA4 Ce_des6 Ce_des5 Ce_PSE1 6PBDHGLA Pt_des6 — Pp_PSE1 7 PBARAI Pt_des6 Pt_des5 Pp_PSE1

[0292] Plasmids 1 to 5 are pUC Derivatives, Plasmids 6 to 7 are BinaryPlant Transformation Vectors

[0293] Pp=Physcomitrella patens, Pt=Phaeodactylum tricornutum

[0294] Pp_PSE1 corresponds to the sequence of SEQ ID NO: 9.

[0295] PSE=PUFA-specific Δ6-elongase

[0296] Ce_des5=Caenorhabditis elegans Δ5-desaturase (Genbank Acc. No.AF078796)

[0297] Ce_des6=Caenorhabditis elegans Δ6-desaturase (Genbank Acc. No.AF031477, bases 11-1342)

[0298] Ce_PSE1=Caenorhabditis elegans Δ6-elongase (Genbank Acc. No.AF244356, bases 1-867)

[0299] Further desaturases or elongase gene sequences can also beinserted into expression cassettes of the above-described type, such as,for example, Genbank Acc. No. AF231981, NM_(—)013402, AF206662,AF268031, AF226273, AF110510 or AF110509.

[0300] iii) Transfer of Expression Cassettes into Vectors for theTransformation of Agrobacterium tumefaciens and for the Transformationof Plants

[0301] Chimeric gene constructs based on those described in pUC19 can beinserted into the binary vector pGPTV by means of AscI. For thispurpose, the multiple cloning sequence is extended by an AscI cleavagesite. For this purpose, the polylinker is newly synthesized as twodouble-stranded oligonucleotides, an additional AscI DNA sequence beinginserted. The oligonucleotide is inserted into the vector PGPTV by meansof EcoRI and HindIII.

[0302] This gives rise to plasmid pGPTV+AscI. The cloning techniquesrequired are known to the skilled worker and can simply be found asdescribed in Example 1.

Example 12 In-vivo Mutagenesis

[0303] The in-vivo mutagenesis of microorganisms can be performed bypassaging the plasmid DNA (or any other vector DNA) via E. coli or othermicroorganisms (for example Bacillus spp. or yeasts such asSaccharomyces cerevisiae) in which the ability of maintaining theintegrity of the genetic information is disrupted. Conventional mutatorstrains have mutations in the genes for the DNA repair system (forexample mutHLS, mutD, mutT and the like; as reference, see Rupp, W. D.(1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, pp.2277-2294, ASM: Washington). These strains are known to the skilledworker. The use of these strains is illustrated for example in Greener,A., and Callahan, M. (1994) Strategies 7:32-34. The transfer of mutatedDNA molecules into plants is preferably effected after themicroorganisms have been selected and tested. Transgenic plants aregenerated in accordance with various examples in the examples section ofthe present document.

Example 13 Studying the Expression of a Recombinant Gene Product in aTransformed Organism

[0304] The activity of a recombinant gene product in the transformedhost organism can be measured at the transcription and/or translationlevel.

[0305] A suitable method for determining the amount of transcription ofthe gene (which indicates the amount of RNA available for translation ofthe gene product) is to carry out a Northern blot as specifiedhereinbelow (for reference, see Ausubel et al. (1988) Current Protocolsin Molecular Biology, Wiley: New York, or the abovementioned examplessection) in which a primer which is designed such that it binds to thegene of interest is labeled with a detectable label (usuallyradioactivity or chemiluminescent) so that, when the total RNA of aculture of the organism is extracted, separated on a gel, transferred toa stable matrix and incubated with this probe, binding and the extent ofbinding of the probe indicate the presence and also the quantity of themRNA for this gene. This information indicates the degree oftranscription of the transformed gene. The total cell RNA can beprepared from cells, tissues or organs by a plurality of methods, all ofwhich are known in the art, such as, for example, the method of Bormann,E. R., et al. (1992) Mol. Microbiol. 6:317-326.

[0306] Northern Hybridization

[0307] For the RNA hybridization, 20 μg of total RNA or 1 μg of poly(A)⁺RNA were separated by gel electrophoresis in 1.25% strength agarose gelusing formaldehyde as described by Amasino (1986, Anal. Biochem. 152,304), transferred to positively charged nylon membranes (Hybond N+,Amersham, Braunschweig) by capillary attraction using 10×SSC,immobilized by means of UV light and prehybridized for 3 hours at 68° C.using hybridization buffer (10% dextran sulfate w/v, 1 M NaCl, 1% SDS,100 mg herring sperm DNA). The DNA probe had been labeled with theHighprime DNA labeling kit (Roche, Mannheim, Germany) during theprehybridization stage using alpha-³²P-dCTP (Amersham, Braunschweig,Germany). The hybridization was carried out after adding the labeled DNAprobe in the same buffer at 68° C. overnight. The wash steps werecarried out twice for 15 minutes using 2×SSC and twice for 30 minutesusing 1×SSC, 1% SDS, at 68° C. The sealed filters were exposed at −70°C. for a period of 1 to 14 days.

[0308] Standard techniques such as a Western blot (see, for example,Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: NewYork) can be employed for studing the presence or the relative quantityof protein translated from this mRNA. In this method, the total cellproteins are extracted, separated by means of gel electrophoresis,transferred to a matrix such as nitrocellulose, and incubated with aprobe such as an antibody which specifically binds to the desiredprotein. This probe is usually provided with a chemiluminescent orcolorimetric label which can be detected readily. The presence and thequantity of the label observed indictes the presence and quantity of thedesired mutated protein which is present in the cell.

Example 14 Analysis of the Effect of the Recombinant Proteins on theProduction of the Desired Product

[0309] The effect of the genetic modification in plants, fungi, algae,ciliates or on the production of a desired compound (such as a fattyacid) can be determined by growing the modified microorganisms or themodified plant under suitable conditions (such as those described above)and analyzing the medium and/or the cell components for the increasedproduction of the desired product (i.e. of lipids or a fatty acid).These analytical techniques are known to the skilled worker andencompass spectroscopy, thin-layer chromatography, various stainingmethods, enzymatic and microbiological methods, and analyticalchromatography such as high-performance liquid chromatography (see, forexample, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp.89-90 and pp. 443-613, V C H Weinheim (1985); Fallon, A., et al., (1987)“Applications of HPLC in Biochemistry” in: Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993)Biotechnology, Vol. 3, Chapter III: “Product recovery and purification”,pp. 469-714, V C H Weinheim; Belter, P. A., et al. (1988)Bioseparations: downstream processing for Biotechnology, John Wiley andSons; Kennedy, J. F., and Cabral, J. M. S. (1992) Recovery processes forbiological Materials, John Wiley and Sons; Shaeiwitz, J. A., and Henry,J. D. (1988) Biochemical Separations, in: Ullmann's Encyclopedia ofIndustrial Chemistry, Vol. B3; Chapter 11, pp. 1-27, V C H Weinheim; andDechow, F. J. (1989) Separation and purification techniques inbiotechnology, Noyes Publications).

[0310] In addition to the abovementioned methods, plant lipids areextracted from plant materials as described by Cahoon et al. (1999)Proc. Natl. Acad. Sci. USA 96 (22):12935-12940, and Browse et al. (1986)Analytic Biochemistry 152:141-145. Qualitative and quantitative lipid orfatty acid analysis is described in Christie, William W., Advances inLipid Methodology, Ayr/Scotland: Oily Press (Oily Press Lipid Library;2); Christie, William W., Gas Chromatography and Lipids. A PracticalGuide—Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 S. (OilyPress Lipid Library; 1); “Progress in Lipid Research, Oxford: PergamonPress, 1 (1952)—16 (1977) under the title: Progress in the Chemistry ofFats and Other Lipids CODEN.

[0311] In addition to measuring the end product of the fermentation, itis also possible to analyze other components of the metabolic pathwayswhich are used for producing the desired compound, such as intermediatesand byproducts, in order to determine the overall production efficiencyof the compound. The analytical methods encompass measurements of thenutrient quantities in the medium (for example sugars, hydrocarbons,nitrogen sources, phosphate and other ions), measurements of biomassconcentration and growth, analysis of the production of customarymetabolites of biosynthetic pathways, and measurements of gases whichare generated during fermentation. Standard methods for thesemeasurements are described in Applied Microbial Physiology; A PracticalApproach, P. M. Rhodes and P. F. Stanbury, Ed., IRL Press, S. 103-129;131-163 and 165-192 (ISBN: 0199635773) and references cited therein.

[0312] One example is the analysis of fatty acids (abbreviations: FAME,fatty acid methyl ester; GC-MS, gas-liquid chromatography/massspectrometry; TAG, triacylglycerol; TLC, thin-layer chromatography).

[0313] The unambiguous detection of the presence of fatty acid productscan be obtained by analyzing recombinant organisms by analyticalstandard methods: GC, GC-MS or TLC, as they are described on severaloccasions by Christie and the references therein (1997, in: Advances onLipid Methodology, Fourth Edition: Christie, Oily Press, Dundee,119-169; 1998, gas chromatography/mass spectrometry methods, Lipide33:343-353).

[0314] The material to be analyzed can be disrupted by ultrasonication,grinding in a glass mill, liquid nitrogen and grinding or by otherapplicable methods. After disruption, the material must be centrifuged.The sediment is resuspended in distilled water, heated for 10 minutes at100° C., ice-cooled and recentrifuged, followed by extraction in 0.5 Msulfuric acid in methanol with 2% dimethoxypropane for 1 hour at 90° C.,which leads to hydrolyzed oil and lipid compounds which givetransmethylated lipids. These fatty acid methyl esters are extracted inpetroleum ether and finally subjected to GC analysis using a capillarycolumn (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 μm, 0.32 mm) at atemperature gradient between 170° C. and 240° C. for 20 minutes and 5minutes at 240° C. The identity of the resulting fatty acid methylesters must be defined using standards which are commercially available(i.e. Sigma).

[0315] In the case of fatty acids for which no standards are available,the identity must be demonstrated via derivatization followed by GC/MSanalysis. For example, the localization of fatty acids with triple bondsmust be demonstrated via GC/MS following derivatization with4,4-dimethoxyoxazolin derivatives (Christie, 1998, see above).

[0316] Expression Constructs in Heterologous Microbial Systems

[0317] Strains, Wash Conditions and Plasmids

[0318] The Escherichia coli strain XL1 Blue MRF′ kan (Stratagene) wasused for subcloning novel Physcomitrella patens desaturasepPDesaturase1. For functionally expressing this gene, we used theSaccharomyces cerevisiae strain INVSc 1 (Invitrogen Co.). E. coli wasgrown in Luria-Bertini broth (LB, Duchefa, Haarlem, the Netherlands) at37° C. If necessary, ampicillin (100 mg/liter) was added, and 1.5% ofagar (w/v) was added for solid LB media. S. cerevisiae was grown at 30°C. either in YPG medium or in complete minimal dropout uracil medium(CMdum; see in: Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D.D., Seidman, J. G., Smith, J. A., Struhl, K., Albright, L. B., Coen, D.M., and Varki, A. (1995) Current Protocols in Molecular Biology, JohnWiley & Sons, New York) together with 2% (w/v) of either raffinose orglucose. For solid media, 2% (w/v) of Bacto™-agar (Difco) were added.The plasmids used for cloning an expression are pUC18 (Pharmacia) andpYES2 (Invitrogen Co.).

Example 16 Cloning and Expression of PUFA-Specific Phaeodactylumtricornutum Desaturases

[0319] For the expression in yeast, the Phaeodactylum tricornutum cDNAclones from SEQ ID NO: 1, 3, 5 or 11 or the sequences from SEQ ID NO: 7or 9 or other desired sequences were first modified in such a way thatonly the coding regions are amplified by means of polymerase chainreaction with the aid of two oligonucleotides. Care was taken that aconsensus sequence for the start codon was retained for efficienttranslation. To this end, either the base sequence ATA or AAA wasselected and inserted into the sequence before the ATG (Kozak, M. (1986)Point mutations define a sequence flanking the AUG initiator codon thatmodulates translation by eukaryotic ribosomes, Cell 44, 283-292). Arestriction cleavage site was additionally introduced before thisconsensus triplet, which restriction cleavage site must be compatiblewith the cleavage site of the target vector into which the fragment isto be cloned and with the aid of which gene expression is to take placein microorganisms or plants.

[0320] The PCR reaction was carried out with plasmid DNA as template ina thermocycler (Biometra) using Pfu DNA (Stratagene) polymerase and thefollowing temperature program: 3 minutes at 96° C., followed by 30cycles with 30 seconds at 96° C., 30 seconds at 55° C. and 2 minutes at72° C., 1 cycle with 10 minutes at 72° C. and stop at 4° C. Theannealing temperature was varied depending on the oligonucleotideschosen. A synthesis time of approximately one minute can be assumed perkilobase pairs of DNA. Further parameters which have an effect on thePCR, such as, for example, Mg ions, salt, DNA polymerase and the likeare known to the skilled worker and can be varied as required.

[0321] The correct size of the amplified DNA fragment was controlled bymeans of agarose TBE gel electrophoresis. The amplified DNA wasextracted from the gel using the QIAquick gel extraction kit (QIAGEN)and ligated into the SmaI restriction site of the dephosphorylatedvector pUC18 using the Sure Clone Ligations Kit (Pharmacia), giving riseto the pUC derivatives. Following the transformation of E. coli XL1 BlueMRF' kan, a DNA mini preparation (Riggs, M. G., & McLachlan, A. (1986) Asimplified screening procedure for large numbers of plasmidmini-preparation. BioTechniques 4, 310-313) was carried out onampicillin-resistant transformants, and positive clones were identifiedby means of BamHI restriction analysis. The sequence of the cloned PCRproduct was confirmed by resequencing using the ABI PRISM Big DyeTerminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer,Weiterstadt).

[0322] Δ5 acyl lipid desaturase, Pt_des5

[0323] Primer 1 GAG CTC ACA TAA TGG CTC CGG ATG CGG ATA AGC

[0324] Primer 2 CTC GAG TTA CGC CCG TCC GGT CAA GGG

[0325] The PCR fragment (1428 bp) was cloned into pUC18 with the aid ofthe Sure Clone kit (Pharmacia), the inserted fragment was digested withSacI/XhoI, and the fragment was inserted into pYES2 or pYES6 with theaid of suitable restriction cleavage sites.

[0326] Δ6 acyl lipid desaturase, Pt_des6

[0327] Primer 3 GGA TCC ACA TAA TGG GCA AAG GAG GGG ACG CTC GGG

[0328] Primer 4 CTC GAG TTA CAT GGC GGG TCC ATC GGG

[0329] The PCR fragment (1451 bp) was cloned into pUC18 with the aid ofthe Sure Clone kit (Pharmacia), the inserted fragment was digested withBamHI/XhoI, and the fragment was inserted into pYES2 or pYES6 with theaid of suitable restriction cleavage sites.

[0330] Δ12 acyl lipid desaturase, Pt_des12

[0331] Primer 5 GGA TCC ACA TAA TGG TTC GCT TTT CAA CAG CC

[0332] Primer 6 CTC GAG TTA TTC GCT CGA TAA TTT GC

[0333] Δ12 acyl lipid desaturase, Pt_des12.2

[0334] Primer 7 GGA TCC ACA TAA TGG GTA AGG GAG GTC AAC G

[0335] Primer 8 CTC GAG TCA TGC GGC TTT GTT TCG C

[0336] The PCR fragment (1505 bp) was cloned into pUC18 with the aid ofthe Sure Clone kit (Pharmacia), the inserted fragment was digested withBamHI/XhoI, and the fragment was inserted into pYES2 or pYES6 with theaid of suitable restriction cleavage sites.

[0337] The plasmid DNA was cleaved with restriction enzyme(s) to matchthe introduced cleavage site of the primer sequence, and the fragmentobtained was ligated into the compatible restriction sites of thedephosphorylated yeast/E. coli shuttle vector pYES2 or pYES6, givingrise to pYES derivatives. Following the transformation of E. coli, andDNA minipreparation from the transformants, the orientation of the DNAfragment in the vector was verified by suitable restriction cleavage orsequencing. One clone was used for the DNA maxipreparation with theNucleobond® AX 500 plasmid DNA extraction kit (Macherey-Nagel,Düringen).

[0338]Saccharomyces cerevisiae INVSc1 was transformed with the pYESderivatives and PYES blank vector by means of a PEG/lithium acetateprotocol (Ausubel et al., 1995). Following selection on CMdum agarplates with 2% glucose, PYES derivative transformants and one pYES2transformant were selected for further cultivation and functionalexpression. For pYES6 derivatives, blasticidin was used asantimetabolite. In the case of coexpression based on pYES2 and pYES6,selection was carried out with blasticidin on minimal medium.

[0339] Functional Expression of a Desaturase Activity in Yeast

[0340] Preculture

[0341] 20 ml of liquid CMdum dropout uracil medium which, however,contains 2% (w/v) of raffinose were inoculated with the transgenic yeastclones (pYES2) and grown for 3 days at 30° C., 200 rpm, until an opticaldensity at 600 nm (OD₆₀₀) of 1.5 to 2 had been reached. If pYES6 wasused as vector, there was additional selection on blasticidin asantimetabolite.

[0342] Main Culture

[0343] For expression, 20 ml of liquid CMdum dropout uracil mediumwhich, however, contains 2% of raffinose and 1% (v/v) of Tergitol NP-40were supplemented with fatty acid substrates to a final concentration of0.003% (w/v). The media were inoculated with the precultures to an OD₆₀₀of 0.05. Expression was induced for 16 hours at an OD₆₀₀ of 0.2, using2% (w/v) of galactose, whereupon the cultures were harvested at an OD₆₀₀of 0.8-1.2.

[0344] Fatty Acid Analysis

[0345] The total fatty acids were extracted from yeast cultures andanalyzed by means of gas chromatography. To this end, cells of 5 ml ofculture were harvested by centrifugation (1 000×g, 10 minutes, 4° C.)and washed once with 100 mM NaHCO₃, pH 8.0 in order to remove residualmedium and fatty acids. To prepare the fatty acid methyl ester(s) (FAMEsor, in the singular, FAME), the cell sediments were treated for 1 hourat 80° C. with 1 M methanolic H₂SO₄ and 2% (v/v) dimethoxypropane. TheFAMEs were extracted twice with 2 ml of petroleum ether, washed oncewith 100 mM NaHCO₃, pH 8.0, and once with distilled water, and driedwith Na₂SO₄. The organic solvent was evaporated under a stream of argon,and the FAMEs were dissolved in 50 μl of petroleum ether. The sampleswere separated on a ZEBRON-ZB Wax capillary column (30 m, 0.32 mm, 0.25μlm; Phenomenex) in a Hewlett Packard 6850 gas chromatograph equippedwith flame ionization detector. The oven temperature was programmed from70° C. (hold for 1 minute) to 200° C. at a rate of 20° C./minute, thento 250° C. (hold for 5 minutes) at a rate of 5° C./minute and finally to260° C. at a rate of 5° C./minute. Nitrogen was used as the carrier gas(4.5 ml/minute at 70° C.). The fatty acids were identified by comparisonwith retention times of FAME standards (SIGMA).

[0346] Expression Analysis

[0347] The ratios of the fatty acid substrates which had been added andtaken up were determined, thus recording the quantity and quality of thedesaturase reaction in accordance with Table 6, Table 7 and Table 8.

[0348] The result of the expression of a Phaeodactylum tricornutumΔ6-acyl lipid desaturase in yeast: TABLE 6 pYES2 pYES2-Ptd6 fed withFatty acid — — +18:2 +18:3 16:0 13.3 18.9 28.4 16.7 16:1Δ9 45.4 44.712.5 16.9 16:2Δ6, 9 — 4.3 — — 18:0 4.9 6.3 10.4 9.1 18:1Δ9 36.4 24.1 6.811.8 18:2Δ6, 9 — 1.8 — — 18:2Δ9, 12 — — 33.4 — 18:3Δ6, 12, 15 — — 4.9 —18:3Δ9, 12, 15 — — 43.1 18:4Δ6, 9, 12, 15 — — — 2.3

[0349] The data represent mol% of corresponding cis-fatty acids.

[0350] Result of the expression of a Phaeodactylum tricornutum Δ5-acyllipid desaturase in yeast: TABLE 7 Fatty pYES2 pYES_PtD5 construct fedwith acid Blank Control 18:2 18:3 20:1Δ8 −20:1Δ11 20:2Δ11,14 20:3Ω320:3Ω6 16:0Δ 16.9 20.4 27.7 24.4 16.2 21 17.6 19.5 22.8 16:1Δ9 44.7 44.113.2 9.6 37.4 39.4 38.3 36.9 30.7 18:0 6.1 6.9 10.54 9.8 4.7 7.9 6.3 6.88.2 18:1Δ9 31.72 28.1 8.77 6 15 26 29.5 25.6 21.1 18:2Δ5,9 0.17 0 0 00.09 0.21 0.09 9 18:2Δ9,12 — 39.7 — — — — — — 18:3Δ9,12,15 — 49.9 -- — —— — 20:1Δ8 — — 25.5 — — — — 20:1Δ11 — — — 5.41 — — — 20:2Δ5,11 — — —0.21 — — — 20:2Δ11,14 — — — — — 6.48 — — 20:3Δ5,11,14 — 0.76 — —20:3Δ11,14,17 — — — — — — 9.83 — 20:3Δ8,11,14 — — — — — — — 13.6920:4Δ5,11,14,17 — — — — — — 1.16 — 20:4Δ5,8,11,14 — — — — — — — 3.08

[0351] The data represent mol% fatty acids of cis-fatty acids.

[0352] Further feeding experiments have revealed that C18:1Δ9 was notdesaturated in the presence of C18:2Δ9,11 or C18:3Δ9,12,15 or C20:1Δ8fatty acids, while C18:1 is also desaturated in the presence ofC20:1Δ11, C20:2Δ11,14 and C20:3Δ8,11,14. Also, no desaturation tookplace in the presence of C20:3Δ8,11,14.

[0353] When using the protease-deficient yeast strain C13BYS86 (Kunze I.et al., Biochemica et Biophysica Acta (1999) 1410:287-298) forexpressing the Phaeodactylum tricornutum Δ5-desaturase on completemedium with blasticidin, it was found that C20:4 Δ8,11,14,17 assubstrate of Δ5-desaturase gave a conversion rate of 20% and was thusequally well converted as C20:3 Δ8,11,14. As an alternative, theauxotrophism markers leu2, ura3 or his can also be used for geneexpression.

[0354] In a further coexpression experiment of PhaeodactylumΔ5-desaturase and Physcomitrella Δ6-elongase, the strain used was UTL7A(Warnecke et al., J. Biol. Chem. (1999) 274(19):13048-13059),Δ5-desaturase converting approximately 10% of C20:3 Δ8,11,14 into C20:4Δ5,8,11,14.

[0355] Further feeding experiment with a wide range of other fatty acidsalone or in combination (for example linoleic acid, 20:3 Δ5,11,14-fattyacid, α- or γ-linolenic acid, stearidonic acid, arachidonic acid,eicosapentaenoic acid and the like) can be carried out for confirmingthe substrate specificity and substrate selectivity of these desaturasesin greater detail. TABLE 8 Result of coexpressing a Phaeodactylumtricornutum Δ5-acyl lipid desaturase and a moss Δ6-elongase in yeastbased on the expression vectors pYES2 and pYES6 pYES2-Elo pYES2-Elo andpYES6-Ptd5 +18:3 +18:4 +18:3 +18:4 16:0 15.0 14.8 15.6 15.1 16:1Δ9 27.729.2 27.5 29.0 18:0 5.6 6.3 5.7 6.4 18:1Δ9 17.1 30.8 27.4 31.6 18:3Δ6,9, 12 7.60 — 7.8 — 18:4Δ6, 9, 12, 15 — 6.71 — 6.4 20:3Δ8, 11, 14 15.92 —13.55 — 20:4Δ5, 8, 11, 14 — — 1.31 — 20:4Δ8, 11, 14, 17 — 11.4 — 10.3120:5Δ5, 8, 11, 14, 17 — — — 0.53

[0356] The substrate conversions reveal that the PhaeodactylumΔ5-desaturase and the Physcomitrella patens Δ6 elongase which were usedare suitable with regard to substrate activity and in particularsubstrate specificity for producing arachidonic acid or eicosapentaenoicacid with the aid of sequences according to the invention.

[0357] The fragmentation patterns and mass spectra of DMOX derivativesof standards and the peak fractions of the fatty acids shown in Tables6, 7 and 8 and identified by GC show, in comparison, identical results,thus confirming the respective position of the double bond beyond simpleGC detection.

Example 17 Purification of the Desired Product from TransformedOrganisms

[0358] The recovery of the desired product from plant material or fungi,algae, ciliates, animal cells or the supernatant of the above-describedcultures can be performed by various methods known in the art. If thedesired product is not secreted from the cells, the cells can beharvested from the culture by low-speed centrifugation, the cells can belysed by standard techniques, such as mechanical force or sonication.Organs of plants can be separated mechanically from other tissue orother organs. Following homogenization, the cell debris is removed bycentrifugation, and the supernatant fraction comprising the solubleproteins is retained for further purification of the desired compounds.If the product is secreted from desired cells, then the cells areremoved from the culture by low-speed centrifugation, and thesupernatant fraction is retained for further purification.

[0359] The supernatant fraction from each purification method issubjected to chromatography with a suitable resin, the desired moleculeeither being retained on the chromatography resin while many of theimpurities of the sample are not, or the impurities being retained bythe resin while the sample is not. These chromatography steps can berepeated if necessary, using the same or different chromatographyresins. The skilled worker is familiar with the selection of suitablechromatography resins and their most effective application for aparticular molecule to be purified. The purified product can beconcentrated by filtration or ultrafiltration, and stored at atemperature at which the stability of the product is maximized.

[0360] A wide spectrum of purification methods is known in the art, andthe above purification method is not intended to be limiting. Thesepurification methods are described, for example, in Bailey, J. E., &Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill: NewYork (1986).

[0361] The identity and purity of the isolated compounds may be assessedby techniques which are standard in the art. They includehigh-performance liquid chromatography (HPLC), spectroscopic methods,staining methods, thin-layer chromatography, in particular thin-layerchromatography and flame ionization detection (IATROSCAN, Iatron, Tokyo,Japan), NIRS, enzyme assays or microbiological tests. Such analyticalmethods are reviewed in: Patek et al. (1994) Appl. Environ. Microbiol.60:133-140; Malakhova et al. (1996) Biotekhnologiya 11:27-32; andSchmidt et al. (1998) Bioprocess Engineer. 19:67-70. Ulmann'sEncyclopedia of Industrial Chemistry (1996) Vol. A27, V C H Weinheim,pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, pp. 575-581 and pp.581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistryand Molecular Biology, John Wiley and Sons; Fallon, A., et al. (1987)Applications of HPLC in Biochemistry in: Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 17.

[0362] Equivalents

[0363] The skilled worker recognizes, or will be able to ascertain, anumber of equivalents of the specific use forms according to theinvention described herein by using no more than routine experiments.These equivalents are intended to be encompassed by the claims.

1 31 1 1652 DNA Phaeodactylum tricornutum CDS (115)..(1524) 1 gacccaacaaacccaacaat cccaacaatc ccatcaacag gaattgggtt tcgttgagtc 60 aataattgctagaatccaaa cagacagaca gagaccaacc gcatctatta caga atg 117 Met 1 gct ccggat gcg gat aag ctt cga caa cgc cag acg act gcg gta gcg 165 Ala Pro AspAla Asp Lys Leu Arg Gln Arg Gln Thr Thr Ala Val Ala 5 10 15 aag cac aatgct gct acc ata tcg acg cag gaa cgc ctt tgc agt ctg 213 Lys His Asn AlaAla Thr Ile Ser Thr Gln Glu Arg Leu Cys Ser Leu 20 25 30 tct tcg ctc aaaggc gaa gaa gtc tgc atc gac gga atc atc tat gac 261 Ser Ser Leu Lys GlyGlu Glu Val Cys Ile Asp Gly Ile Ile Tyr Asp 35 40 45 ctc caa tca ttc gatcat ccc ggg ggt gaa acg atc aaa atg ttt ggt 309 Leu Gln Ser Phe Asp HisPro Gly Gly Glu Thr Ile Lys Met Phe Gly 50 55 60 65 ggc aac gat gtc actgta cag tac aag atg att cac ccg tac cat acc 357 Gly Asn Asp Val Thr ValGln Tyr Lys Met Ile His Pro Tyr His Thr 70 75 80 gag aag cat ttg gaa aagatg aag cgt gtc ggc aag gtg acg gat ttc 405 Glu Lys His Leu Glu Lys MetLys Arg Val Gly Lys Val Thr Asp Phe 85 90 95 gtc tgc gag tac aag ttc gatacc gaa ttt gaa cgc gaa atc aaa cga 453 Val Cys Glu Tyr Lys Phe Asp ThrGlu Phe Glu Arg Glu Ile Lys Arg 100 105 110 gaa gtc ttc aag att gtg cgacga ggc aag gat ttc ggt act ttg gga 501 Glu Val Phe Lys Ile Val Arg ArgGly Lys Asp Phe Gly Thr Leu Gly 115 120 125 tgg ttc ttc cgt gcg ttt tgctac att gcc att ttc ttc tac ctg cag 549 Trp Phe Phe Arg Ala Phe Cys TyrIle Ala Ile Phe Phe Tyr Leu Gln 130 135 140 145 tac cat tgg gtc acc acggga acc tct tgg ctg ctg gcc gtg gcc tac 597 Tyr His Trp Val Thr Thr GlyThr Ser Trp Leu Leu Ala Val Ala Tyr 150 155 160 gga atc tcc caa gcg atgatt ggc atg aat gtc cag cac gat gcc aac 645 Gly Ile Ser Gln Ala Met IleGly Met Asn Val Gln His Asp Ala Asn 165 170 175 cac ggg gcc acc tcc aagcgt ccc tgg gtc aac gac atg cta ggc ctc 693 His Gly Ala Thr Ser Lys ArgPro Trp Val Asn Asp Met Leu Gly Leu 180 185 190 ggt gcg gat ttt att ggtggt tcc aag tgg ctc tgg cag gaa caa cac 741 Gly Ala Asp Phe Ile Gly GlySer Lys Trp Leu Trp Gln Glu Gln His 195 200 205 tgg acc cac cac gct tacacc aat cac gcc gag atg gat ccc gat agc 789 Trp Thr His His Ala Tyr ThrAsn His Ala Glu Met Asp Pro Asp Ser 210 215 220 225 ttt ggt gcc gaa ccaatg ctc cta ttc aac gac tat ccc ttg gat cat 837 Phe Gly Ala Glu Pro MetLeu Leu Phe Asn Asp Tyr Pro Leu Asp His 230 235 240 ccc gct cgt acc tggcta cat cgc ttt caa gca ttc ttt tac atg ccc 885 Pro Ala Arg Thr Trp LeuHis Arg Phe Gln Ala Phe Phe Tyr Met Pro 245 250 255 gtc ttg gct gga tactgg ttg tcc gct gtc ttc aat cca caa att ctt 933 Val Leu Ala Gly Tyr TrpLeu Ser Ala Val Phe Asn Pro Gln Ile Leu 260 265 270 gac ctc cag caa cgcggc gca ctt tcc gtc ggt atc cgt ctc gac aac 981 Asp Leu Gln Gln Arg GlyAla Leu Ser Val Gly Ile Arg Leu Asp Asn 275 280 285 gct ttc att cac tcgcga cgc aag tat gcg gtt ttc tgg cgg gct gtg 1029 Ala Phe Ile His Ser ArgArg Lys Tyr Ala Val Phe Trp Arg Ala Val 290 295 300 305 tac att gcg gtgaac gtg att gct ccg ttt tac aca aac tcc ggc ctc 1077 Tyr Ile Ala Val AsnVal Ile Ala Pro Phe Tyr Thr Asn Ser Gly Leu 310 315 320 gaa tgg tcc tggcgt gtc ttt gga aac atc atg ctc atg ggt gtg gcg 1125 Glu Trp Ser Trp ArgVal Phe Gly Asn Ile Met Leu Met Gly Val Ala 325 330 335 gaa tcg ctc gcgctg gcg gtc ctg ttt tcg ttg tcg cac aat ttc gaa 1173 Glu Ser Leu Ala LeuAla Val Leu Phe Ser Leu Ser His Asn Phe Glu 340 345 350 tcc gcg gat cgcgat ccg acc gcc cca ctg aaa aag acg gga gaa cca 1221 Ser Ala Asp Arg AspPro Thr Ala Pro Leu Lys Lys Thr Gly Glu Pro 355 360 365 gtc gac tgg ttcaag aca cag gtc gaa act tcc tgc act tac ggt gga 1269 Val Asp Trp Phe LysThr Gln Val Glu Thr Ser Cys Thr Tyr Gly Gly 370 375 380 385 ttc ctt tccggt tgc ttc acg gga ggt ctc aac ttt cag gtt gaa cac 1317 Phe Leu Ser GlyCys Phe Thr Gly Gly Leu Asn Phe Gln Val Glu His 390 395 400 cac ttg ttccca cgc atg agc agc gct tgg tat ccc tac att gcc ccc 1365 His Leu Phe ProArg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala Pro 405 410 415 aag gtc cgcgaa att tgc gcc aaa cac ggc gtc cac tac gcc tac tac 1413 Lys Val Arg GluIle Cys Ala Lys His Gly Val His Tyr Ala Tyr Tyr 420 425 430 ccg tgg atccac caa aac ttt ctc tcc acc gtc cgc tac atg cac gcg 1461 Pro Trp Ile HisGln Asn Phe Leu Ser Thr Val Arg Tyr Met His Ala 435 440 445 gcc ggg accggt gcc aac tgg cgc cag atg gcc aga gaa aat ccc ttg 1509 Ala Gly Thr GlyAla Asn Trp Arg Gln Met Ala Arg Glu Asn Pro Leu 450 455 460 465 acc ggacgg gcg taa aagtacacga cacgaccaaa ggtggcgtat ggtgatctct 1564 Thr Gly ArgAla agaaaacaga catagcctac tggaaatatc gacgtccaaa caataatttt aaagactatt1624 tttctgcgta aaaaaaaaaa aaaaaaaa 1652 2 469 PRT Phaeodactylumtricornutum 2 Met Ala Pro Asp Ala Asp Lys Leu Arg Gln Arg Gln Thr ThrAla Val 1 5 10 15 Ala Lys His Asn Ala Ala Thr Ile Ser Thr Gln Glu ArgLeu Cys Ser 20 25 30 Leu Ser Ser Leu Lys Gly Glu Glu Val Cys Ile Asp GlyIle Ile Tyr 35 40 45 Asp Leu Gln Ser Phe Asp His Pro Gly Gly Glu Thr IleLys Met Phe 50 55 60 Gly Gly Asn Asp Val Thr Val Gln Tyr Lys Met Ile HisPro Tyr His 65 70 75 80 Thr Glu Lys His Leu Glu Lys Met Lys Arg Val GlyLys Val Thr Asp 85 90 95 Phe Val Cys Glu Tyr Lys Phe Asp Thr Glu Phe GluArg Glu Ile Lys 100 105 110 Arg Glu Val Phe Lys Ile Val Arg Arg Gly LysAsp Phe Gly Thr Leu 115 120 125 Gly Trp Phe Phe Arg Ala Phe Cys Tyr IleAla Ile Phe Phe Tyr Leu 130 135 140 Gln Tyr His Trp Val Thr Thr Gly ThrSer Trp Leu Leu Ala Val Ala 145 150 155 160 Tyr Gly Ile Ser Gln Ala MetIle Gly Met Asn Val Gln His Asp Ala 165 170 175 Asn His Gly Ala Thr SerLys Arg Pro Trp Val Asn Asp Met Leu Gly 180 185 190 Leu Gly Ala Asp PheIle Gly Gly Ser Lys Trp Leu Trp Gln Glu Gln 195 200 205 His Trp Thr HisHis Ala Tyr Thr Asn His Ala Glu Met Asp Pro Asp 210 215 220 Ser Phe GlyAla Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Asp 225 230 235 240 HisPro Ala Arg Thr Trp Leu His Arg Phe Gln Ala Phe Phe Tyr Met 245 250 255Pro Val Leu Ala Gly Tyr Trp Leu Ser Ala Val Phe Asn Pro Gln Ile 260 265270 Leu Asp Leu Gln Gln Arg Gly Ala Leu Ser Val Gly Ile Arg Leu Asp 275280 285 Asn Ala Phe Ile His Ser Arg Arg Lys Tyr Ala Val Phe Trp Arg Ala290 295 300 Val Tyr Ile Ala Val Asn Val Ile Ala Pro Phe Tyr Thr Asn SerGly 305 310 315 320 Leu Glu Trp Ser Trp Arg Val Phe Gly Asn Ile Met LeuMet Gly Val 325 330 335 Ala Glu Ser Leu Ala Leu Ala Val Leu Phe Ser LeuSer His Asn Phe 340 345 350 Glu Ser Ala Asp Arg Asp Pro Thr Ala Pro LeuLys Lys Thr Gly Glu 355 360 365 Pro Val Asp Trp Phe Lys Thr Gln Val GluThr Ser Cys Thr Tyr Gly 370 375 380 Gly Phe Leu Ser Gly Cys Phe Thr GlyGly Leu Asn Phe Gln Val Glu 385 390 395 400 His His Leu Phe Pro Arg MetSer Ser Ala Trp Tyr Pro Tyr Ile Ala 405 410 415 Pro Lys Val Arg Glu IleCys Ala Lys His Gly Val His Tyr Ala Tyr 420 425 430 Tyr Pro Trp Ile HisGln Asn Phe Leu Ser Thr Val Arg Tyr Met His 435 440 445 Ala Ala Gly ThrGly Ala Asn Trp Arg Gln Met Ala Arg Glu Asn Pro 450 455 460 Leu Thr GlyArg Ala 465 3 1434 DNA Phaeodactylum tricornutum CDS (1)..(1434) 3 atgggc aaa gga ggg gac gct cgg gcc tcg aag ggc tca acg gcg gct 48 Met GlyLys Gly Gly Asp Ala Arg Ala Ser Lys Gly Ser Thr Ala Ala 1 5 10 15 cgcaag atc agt tgg cag gaa gtc aag acc cac gcg tct ccg gag gac 96 Arg LysIle Ser Trp Gln Glu Val Lys Thr His Ala Ser Pro Glu Asp 20 25 30 gcc tggatc att cac tcc aat aag gtc tac gac gtg tcc aac tgg cac 144 Ala Trp IleIle His Ser Asn Lys Val Tyr Asp Val Ser Asn Trp His 35 40 45 gaa cat cccgga ggc gcc gtc att ttc acg cac gcc ggt gac gac atg 192 Glu His Pro GlyGly Ala Val Ile Phe Thr His Ala Gly Asp Asp Met 50 55 60 acg gac att ttcgct gcc ttt cac gca ccc gga tcg cag tcg ctc atg 240 Thr Asp Ile Phe AlaAla Phe His Ala Pro Gly Ser Gln Ser Leu Met 65 70 75 80 aag aag ttc tacatt ggc gaa ttg ctc ccg gaa acc acc ggc aag gag 288 Lys Lys Phe Tyr IleGly Glu Leu Leu Pro Glu Thr Thr Gly Lys Glu 85 90 95 ccg cag caa atc gccttt gaa aag ggc tac cgc gat ctg cgc tcc aaa 336 Pro Gln Gln Ile Ala PheGlu Lys Gly Tyr Arg Asp Leu Arg Ser Lys 100 105 110 ctc atc atg atg ggcatg ttc aag tcc aac aag tgg ttc tac gtc tac 384 Leu Ile Met Met Gly MetPhe Lys Ser Asn Lys Trp Phe Tyr Val Tyr 115 120 125 aag tgc ctc agc aacatg gcc att tgg gcc gcc gcc tgt gct ctc gtc 432 Lys Cys Leu Ser Asn MetAla Ile Trp Ala Ala Ala Cys Ala Leu Val 130 135 140 ttt tac tcg gac cgcttc tgg gta cac ctg gcc agc gcc gtc atg ctg 480 Phe Tyr Ser Asp Arg PheTrp Val His Leu Ala Ser Ala Val Met Leu 145 150 155 160 gga aca ttc tttcag cag tcg gga tgg ttg gca cac gac ttt ctg cac 528 Gly Thr Phe Phe GlnGln Ser Gly Trp Leu Ala His Asp Phe Leu His 165 170 175 cac cag gtc ttcacc aag cgc aag cac ggg gat ctc gga gga ctc ttt 576 His Gln Val Phe ThrLys Arg Lys His Gly Asp Leu Gly Gly Leu Phe 180 185 190 tgg ggg aac ctcatg cag ggt tac tcc gta cag tgg tgg aaa aac aag 624 Trp Gly Asn Leu MetGln Gly Tyr Ser Val Gln Trp Trp Lys Asn Lys 195 200 205 cac aac gga caccac gcc gtc ccc aac ctc cac tgc tcc tcc gca gtc 672 His Asn Gly His HisAla Val Pro Asn Leu His Cys Ser Ser Ala Val 210 215 220 gcg caa gat ggggac ccg gac atc gat acc atg ccc ctt ctc gcc tgg 720 Ala Gln Asp Gly AspPro Asp Ile Asp Thr Met Pro Leu Leu Ala Trp 225 230 235 240 tcc gtc cagcaa gcc cag tct tac cgg gaa ctc caa gcc gac gga aag 768 Ser Val Gln GlnAla Gln Ser Tyr Arg Glu Leu Gln Ala Asp Gly Lys 245 250 255 gat tcg ggtttg gtc aag ttc atg atc cgt aac caa tcc tac ttt tac 816 Asp Ser Gly LeuVal Lys Phe Met Ile Arg Asn Gln Ser Tyr Phe Tyr 260 265 270 ttt ccc atcttg ttg ctc gcc cgc ctg tcg tgg ttg aac gag tcc ttc 864 Phe Pro Ile LeuLeu Leu Ala Arg Leu Ser Trp Leu Asn Glu Ser Phe 275 280 285 aag tgc gccttt ggg ctt gga gct gcg tcg gag aac gct gct ctc gaa 912 Lys Cys Ala PheGly Leu Gly Ala Ala Ser Glu Asn Ala Ala Leu Glu 290 295 300 ctc aag gccaag ggt ctt cag tac ccc ctt ttg gaa aag gct ggc atc 960 Leu Lys Ala LysGly Leu Gln Tyr Pro Leu Leu Glu Lys Ala Gly Ile 305 310 315 320 ctg ctgcac tac gct tgg atg ctt aca gtt tcg tcc ggc ttt gga cgc 1008 Leu Leu HisTyr Ala Trp Met Leu Thr Val Ser Ser Gly Phe Gly Arg 325 330 335 ttc tcgttc gcg tac acc gca ttt tac ttt cta acc gcg acc gcg tcc 1056 Phe Ser PheAla Tyr Thr Ala Phe Tyr Phe Leu Thr Ala Thr Ala Ser 340 345 350 tgt ggattc ttg ctc gcc att gtc ttt ggc ctc ggc cac aac ggc atg 1104 Cys Gly PheLeu Leu Ala Ile Val Phe Gly Leu Gly His Asn Gly Met 355 360 365 gcc acctac aat gcc gac gcc cgt ccg gac ttc tgg aag ctc caa gtc 1152 Ala Thr TyrAsn Ala Asp Ala Arg Pro Asp Phe Trp Lys Leu Gln Val 370 375 380 acc acgact cgc aac gtc acg ggc gga cac ggt ttc ccc caa gcc ttt 1200 Thr Thr ThrArg Asn Val Thr Gly Gly His Gly Phe Pro Gln Ala Phe 385 390 395 400 gtcgac tgg ttc tgt ggt ggc ctc cag tac caa gtc gac cac cac tta 1248 Val AspTrp Phe Cys Gly Gly Leu Gln Tyr Gln Val Asp His His Leu 405 410 415 ttcccc agc ctg ccc cga cac aat ctg gcc aag aca cac gca ctg gtc 1296 Phe ProSer Leu Pro Arg His Asn Leu Ala Lys Thr His Ala Leu Val 420 425 430 gaatcg ttc tgc aag gag tgg ggt gtc cag tac cac gaa gcc gac ctt 1344 Glu SerPhe Cys Lys Glu Trp Gly Val Gln Tyr His Glu Ala Asp Leu 435 440 445 gtggac ggg acc atg gaa gtc ttg cac cat ttg ggc agc gtg gcc ggc 1392 Val AspGly Thr Met Glu Val Leu His His Leu Gly Ser Val Ala Gly 450 455 460 gaattc gtc gtg gat ttt gta cgc gat gga ccc gcc atg taa 1434 Glu Phe Val ValAsp Phe Val Arg Asp Gly Pro Ala Met 465 470 475 4 477 PRT Phaeodactylumtricornutum 4 Met Gly Lys Gly Gly Asp Ala Arg Ala Ser Lys Gly Ser ThrAla Ala 1 5 10 15 Arg Lys Ile Ser Trp Gln Glu Val Lys Thr His Ala SerPro Glu Asp 20 25 30 Ala Trp Ile Ile His Ser Asn Lys Val Tyr Asp Val SerAsn Trp His 35 40 45 Glu His Pro Gly Gly Ala Val Ile Phe Thr His Ala GlyAsp Asp Met 50 55 60 Thr Asp Ile Phe Ala Ala Phe His Ala Pro Gly Ser GlnSer Leu Met 65 70 75 80 Lys Lys Phe Tyr Ile Gly Glu Leu Leu Pro Glu ThrThr Gly Lys Glu 85 90 95 Pro Gln Gln Ile Ala Phe Glu Lys Gly Tyr Arg AspLeu Arg Ser Lys 100 105 110 Leu Ile Met Met Gly Met Phe Lys Ser Asn LysTrp Phe Tyr Val Tyr 115 120 125 Lys Cys Leu Ser Asn Met Ala Ile Trp AlaAla Ala Cys Ala Leu Val 130 135 140 Phe Tyr Ser Asp Arg Phe Trp Val HisLeu Ala Ser Ala Val Met Leu 145 150 155 160 Gly Thr Phe Phe Gln Gln SerGly Trp Leu Ala His Asp Phe Leu His 165 170 175 His Gln Val Phe Thr LysArg Lys His Gly Asp Leu Gly Gly Leu Phe 180 185 190 Trp Gly Asn Leu MetGln Gly Tyr Ser Val Gln Trp Trp Lys Asn Lys 195 200 205 His Asn Gly HisHis Ala Val Pro Asn Leu His Cys Ser Ser Ala Val 210 215 220 Ala Gln AspGly Asp Pro Asp Ile Asp Thr Met Pro Leu Leu Ala Trp 225 230 235 240 SerVal Gln Gln Ala Gln Ser Tyr Arg Glu Leu Gln Ala Asp Gly Lys 245 250 255Asp Ser Gly Leu Val Lys Phe Met Ile Arg Asn Gln Ser Tyr Phe Tyr 260 265270 Phe Pro Ile Leu Leu Leu Ala Arg Leu Ser Trp Leu Asn Glu Ser Phe 275280 285 Lys Cys Ala Phe Gly Leu Gly Ala Ala Ser Glu Asn Ala Ala Leu Glu290 295 300 Leu Lys Ala Lys Gly Leu Gln Tyr Pro Leu Leu Glu Lys Ala GlyIle 305 310 315 320 Leu Leu His Tyr Ala Trp Met Leu Thr Val Ser Ser GlyPhe Gly Arg 325 330 335 Phe Ser Phe Ala Tyr Thr Ala Phe Tyr Phe Leu ThrAla Thr Ala Ser 340 345 350 Cys Gly Phe Leu Leu Ala Ile Val Phe Gly LeuGly His Asn Gly Met 355 360 365 Ala Thr Tyr Asn Ala Asp Ala Arg Pro AspPhe Trp Lys Leu Gln Val 370 375 380 Thr Thr Thr Arg Asn Val Thr Gly GlyHis Gly Phe Pro Gln Ala Phe 385 390 395 400 Val Asp Trp Phe Cys Gly GlyLeu Gln Tyr Gln Val Asp His His Leu 405 410 415 Phe Pro Ser Leu Pro ArgHis Asn Leu Ala Lys Thr His Ala Leu Val 420 425 430 Glu Ser Phe Cys LysGlu Trp Gly Val Gln Tyr His Glu Ala Asp Leu 435 440 445 Val Asp Gly ThrMet Glu Val Leu His His Leu Gly Ser Val Ala Gly 450 455 460 Glu Phe ValVal Asp Phe Val Arg Asp Gly Pro Ala Met 465 470 475 5 1651 DNAPhaeodactylum tricornutum CDS (67)..(1554) 5 gaagaaggaa catataaaagtaagccatct cctcggcacc atctaaagac ctaatatcta 60 ctcgtc atg gtt cgc ttttca aca gcc gct cta ctt tct ctg tcg aca 108 Met Val Arg Phe Ser Thr AlaAla Leu Leu Ser Leu Ser Thr 1 5 10 ttg aca act tca tgt att ggt gcc ttccag ctg tct tcg cca gca caa 156 Leu Thr Thr Ser Cys Ile Gly Ala Phe GlnLeu Ser Ser Pro Ala Gln 15 20 25 30 ctt ccg aca agt agg ctt cgt cgg catacg aac acg gcg ccg ctt tcg 204 Leu Pro Thr Ser Arg Leu Arg Arg His ThrAsn Thr Ala Pro Leu Ser 35 40 45 gcc gtg gcc gtc gac tcc ggt tct tcc gatccg gcc ttg gta ggc aac 252 Ala Val Ala Val Asp Ser Gly Ser Ser Asp ProAla Leu Val Gly Asn 50 55 60 ctc ccc ctt ccc aac aac aat gat aat gag gacaag aac cgt aga atg 300 Leu Pro Leu Pro Asn Asn Asn Asp Asn Glu Asp LysAsn Arg Arg Met 65 70 75 cca atg atg gac ttg aaa ggt att gct ctg tct ggtctc aaa ggg caa 348 Pro Met Met Asp Leu Lys Gly Ile Ala Leu Ser Gly LeuLys Gly Gln 80 85 90 gct ctt tcc gtc cga gcg gaa gat ttt cct cag gcg aaagac ttg cgt 396 Ala Leu Ser Val Arg Ala Glu Asp Phe Pro Gln Ala Lys AspLeu Arg 95 100 105 110 gcc gtc att ccg aaa gat tgc ttc gaa ccc gac acggcc aaa tcg ttg 444 Ala Val Ile Pro Lys Asp Cys Phe Glu Pro Asp Thr AlaLys Ser Leu 115 120 125 gga tat ctt tcc gtt tca act atg ggg aca att ctctgc tcc gtc gtc 492 Gly Tyr Leu Ser Val Ser Thr Met Gly Thr Ile Leu CysSer Val Val 130 135 140 ggc gcg aac ctc ctt agt gtg ctc gat ccc tcc aatcca tta acc tgg 540 Gly Ala Asn Leu Leu Ser Val Leu Asp Pro Ser Asn ProLeu Thr Trp 145 150 155 cct ctc tgg gcg gcc tac ggt gcc gtc acg ggg acggtc gcc atg ggg 588 Pro Leu Trp Ala Ala Tyr Gly Ala Val Thr Gly Thr ValAla Met Gly 160 165 170 ctt tgg gtg ctg gcc cac gaa tgc gga cac ggc gccttt tcc aaa aac 636 Leu Trp Val Leu Ala His Glu Cys Gly His Gly Ala PheSer Lys Asn 175 180 185 190 cga tcc ctc cag gat gcc gtg ggg tac att atccat tcc atc atg ctg 684 Arg Ser Leu Gln Asp Ala Val Gly Tyr Ile Ile HisSer Ile Met Leu 195 200 205 gtg cca tac ttt agt tgg cag cga tcg cat gccgtg cat cac cag tat 732 Val Pro Tyr Phe Ser Trp Gln Arg Ser His Ala ValHis His Gln Tyr 210 215 220 acc aat cat atg gaa ctg ggg gaa aca cac gttcct gat cga gcc gat 780 Thr Asn His Met Glu Leu Gly Glu Thr His Val ProAsp Arg Ala Asp 225 230 235 aag gag ggc gag aag agc ctg gcg ctc cgc cagttc atg ttg gat tcc 828 Lys Glu Gly Glu Lys Ser Leu Ala Leu Arg Gln PheMet Leu Asp Ser 240 245 250 ttt ggt aaa gac aag ggc atg aaa gca tac ggaggc ctc cag tcg ttt 876 Phe Gly Lys Asp Lys Gly Met Lys Ala Tyr Gly GlyLeu Gln Ser Phe 255 260 265 270 ttg cat ctc atc gtg gga tgg cca gcc tacctc ctg atc ggt gcg acc 924 Leu His Leu Ile Val Gly Trp Pro Ala Tyr LeuLeu Ile Gly Ala Thr 275 280 285 ggt gga ccc gac cgt ggt atg acc aac catttt tat ccc aac cct ttg 972 Gly Gly Pro Asp Arg Gly Met Thr Asn His PheTyr Pro Asn Pro Leu 290 295 300 tcg acg cca aca cag ccc aag aaa gaa cttttc cct ggg aac tgg aaa 1020 Ser Thr Pro Thr Gln Pro Lys Lys Glu Leu PhePro Gly Asn Trp Lys 305 310 315 gaa aag gtc tac cag tca gat att gga atcgcc gcc gtt gtc ggc gcc 1068 Glu Lys Val Tyr Gln Ser Asp Ile Gly Ile AlaAla Val Val Gly Ala 320 325 330 ctc att gct tgg acc gcc act tcg ggt ctagcc ccc gtc atg gcc ttg 1116 Leu Ile Ala Trp Thr Ala Thr Ser Gly Leu AlaPro Val Met Ala Leu 335 340 345 350 tac ggt ggt ccc ttg atc gtc att aatgcc tgg ctg gta ctg tac acg 1164 Tyr Gly Gly Pro Leu Ile Val Ile Asn AlaTrp Leu Val Leu Tyr Thr 355 360 365 tgg ttg caa cat aca gat acc gat gttccg cac ttt tcc tcc gac aac 1212 Trp Leu Gln His Thr Asp Thr Asp Val ProHis Phe Ser Ser Asp Asn 370 375 380 cac aac ttt gtc aag ggc gca ctg catacg atc gat cgt ccc tac gac 1260 His Asn Phe Val Lys Gly Ala Leu His ThrIle Asp Arg Pro Tyr Asp 385 390 395 aaa ctt gat ccc tgg gga atc ata gacttt ctg cac cac aag att gga 1308 Lys Leu Asp Pro Trp Gly Ile Ile Asp PheLeu His His Lys Ile Gly 400 405 410 aca acg cat gtg gca cac cat ttt gacagt act atc ccc cac tat aag 1356 Thr Thr His Val Ala His His Phe Asp SerThr Ile Pro His Tyr Lys 415 420 425 430 gct cag att gct acc gat gcc atcaaa gcc aag ttt cca gaa gtg tac 1404 Ala Gln Ile Ala Thr Asp Ala Ile LysAla Lys Phe Pro Glu Val Tyr 435 440 445 ctc tat gac ccg aca cca att ccacaa gcc atg tgg cgc gtc gcc aag 1452 Leu Tyr Asp Pro Thr Pro Ile Pro GlnAla Met Trp Arg Val Ala Lys 450 455 460 gga tgt act gca gta gag caa cgcggt gac gcc tgg gtg tgg aaa aac 1500 Gly Cys Thr Ala Val Glu Gln Arg GlyAsp Ala Trp Val Trp Lys Asn 465 470 475 gaa gga ata gaa gat ttg gtg gaacat cgt caa agc aaa tta tcg agc 1548 Glu Gly Ile Glu Asp Leu Val Glu HisArg Gln Ser Lys Leu Ser Ser 480 485 490 gaa taa agcaacatat cgctttatggaagaacaaac gtccattgtg taaaaccctg 1604 Glu 495 ataatttcaa tattgtgttttgttttaaaa aaaaaaaaaa aaaaaaa 1651 6 495 PRT Phaeodactylum tricornutum 6Met Val Arg Phe Ser Thr Ala Ala Leu Leu Ser Leu Ser Thr Leu Thr 1 5 1015 Thr Ser Cys Ile Gly Ala Phe Gln Leu Ser Ser Pro Ala Gln Leu Pro 20 2530 Thr Ser Arg Leu Arg Arg His Thr Asn Thr Ala Pro Leu Ser Ala Val 35 4045 Ala Val Asp Ser Gly Ser Ser Asp Pro Ala Leu Val Gly Asn Leu Pro 50 5560 Leu Pro Asn Asn Asn Asp Asn Glu Asp Lys Asn Arg Arg Met Pro Met 65 7075 80 Met Asp Leu Lys Gly Ile Ala Leu Ser Gly Leu Lys Gly Gln Ala Leu 8590 95 Ser Val Arg Ala Glu Asp Phe Pro Gln Ala Lys Asp Leu Arg Ala Val100 105 110 Ile Pro Lys Asp Cys Phe Glu Pro Asp Thr Ala Lys Ser Leu GlyTyr 115 120 125 Leu Ser Val Ser Thr Met Gly Thr Ile Leu Cys Ser Val ValGly Ala 130 135 140 Asn Leu Leu Ser Val Leu Asp Pro Ser Asn Pro Leu ThrTrp Pro Leu 145 150 155 160 Trp Ala Ala Tyr Gly Ala Val Thr Gly Thr ValAla Met Gly Leu Trp 165 170 175 Val Leu Ala His Glu Cys Gly His Gly AlaPhe Ser Lys Asn Arg Ser 180 185 190 Leu Gln Asp Ala Val Gly Tyr Ile IleHis Ser Ile Met Leu Val Pro 195 200 205 Tyr Phe Ser Trp Gln Arg Ser HisAla Val His His Gln Tyr Thr Asn 210 215 220 His Met Glu Leu Gly Glu ThrHis Val Pro Asp Arg Ala Asp Lys Glu 225 230 235 240 Gly Glu Lys Ser LeuAla Leu Arg Gln Phe Met Leu Asp Ser Phe Gly 245 250 255 Lys Asp Lys GlyMet Lys Ala Tyr Gly Gly Leu Gln Ser Phe Leu His 260 265 270 Leu Ile ValGly Trp Pro Ala Tyr Leu Leu Ile Gly Ala Thr Gly Gly 275 280 285 Pro AspArg Gly Met Thr Asn His Phe Tyr Pro Asn Pro Leu Ser Thr 290 295 300 ProThr Gln Pro Lys Lys Glu Leu Phe Pro Gly Asn Trp Lys Glu Lys 305 310 315320 Val Tyr Gln Ser Asp Ile Gly Ile Ala Ala Val Val Gly Ala Leu Ile 325330 335 Ala Trp Thr Ala Thr Ser Gly Leu Ala Pro Val Met Ala Leu Tyr Gly340 345 350 Gly Pro Leu Ile Val Ile Asn Ala Trp Leu Val Leu Tyr Thr TrpLeu 355 360 365 Gln His Thr Asp Thr Asp Val Pro His Phe Ser Ser Asp AsnHis Asn 370 375 380 Phe Val Lys Gly Ala Leu His Thr Ile Asp Arg Pro TyrAsp Lys Leu 385 390 395 400 Asp Pro Trp Gly Ile Ile Asp Phe Leu His HisLys Ile Gly Thr Thr 405 410 415 His Val Ala His His Phe Asp Ser Thr IlePro His Tyr Lys Ala Gln 420 425 430 Ile Ala Thr Asp Ala Ile Lys Ala LysPhe Pro Glu Val Tyr Leu Tyr 435 440 445 Asp Pro Thr Pro Ile Pro Gln AlaMet Trp Arg Val Ala Lys Gly Cys 450 455 460 Thr Ala Val Glu Gln Arg GlyAsp Ala Trp Val Trp Lys Asn Glu Gly 465 470 475 480 Ile Glu Asp Leu ValGlu His Arg Gln Ser Lys Leu Ser Ser Glu 485 490 495 7 1578 DNAPhyscomitrella patens CDS (1)..(1578) 7 atg gta ttc gcg ggc ggt gga cttcag cag ggc tct ctc gaa gaa aac 48 Met Val Phe Ala Gly Gly Gly Leu GlnGln Gly Ser Leu Glu Glu Asn 1 5 10 15 atc gac gtc gag cac att gcc agtatg tct ctc ttc agc gac ttc ttc 96 Ile Asp Val Glu His Ile Ala Ser MetSer Leu Phe Ser Asp Phe Phe 20 25 30 agt tat gtg tct tca act gtt ggt tcgtgg agc gta cac agt ata caa 144 Ser Tyr Val Ser Ser Thr Val Gly Ser TrpSer Val His Ser Ile Gln 35 40 45 cct ttg aag cgc ctg acg agt aag aag cgtgtt tcg gaa agc gct gcc 192 Pro Leu Lys Arg Leu Thr Ser Lys Lys Arg ValSer Glu Ser Ala Ala 50 55 60 gtg caa tgt ata tca gct gaa gtt cag aga aattcg agt acc cag gga 240 Val Gln Cys Ile Ser Ala Glu Val Gln Arg Asn SerSer Thr Gln Gly 65 70 75 80 act gcg gag gca ctc gca gaa tca gtc gtg aagccc acg aga cga agg 288 Thr Ala Glu Ala Leu Ala Glu Ser Val Val Lys ProThr Arg Arg Arg 85 90 95 tca tct cag tgg aag aag tcg aca cac ccc cta tcagaa gta gca gta 336 Ser Ser Gln Trp Lys Lys Ser Thr His Pro Leu Ser GluVal Ala Val 100 105 110 cac aac aag cca agc gat tgc tgg att gtt gta aaaaac aag gtg tat 384 His Asn Lys Pro Ser Asp Cys Trp Ile Val Val Lys AsnLys Val Tyr 115 120 125 gat gtt tcc aat ttt gcg gac gag cat ccc gga ggatca gtt att agt 432 Asp Val Ser Asn Phe Ala Asp Glu His Pro Gly Gly SerVal Ile Ser 130 135 140 act tat ttt gga cga gac ggc aca gat gtt ttc tctagt ttt cat gca 480 Thr Tyr Phe Gly Arg Asp Gly Thr Asp Val Phe Ser SerPhe His Ala 145 150 155 160 gct tct aca tgg aaa att ctt caa gac ttt tacatt ggt gac gtg gag 528 Ala Ser Thr Trp Lys Ile Leu Gln Asp Phe Tyr IleGly Asp Val Glu 165 170 175 agg gtg gag ccg act cca gag ctg ctg aaa gatttc cga gaa atg aga 576 Arg Val Glu Pro Thr Pro Glu Leu Leu Lys Asp PheArg Glu Met Arg 180 185 190 gct ctt ttc ctg agg gag caa ctt ttc aaa agttcg aaa ttg tac tat 624 Ala Leu Phe Leu Arg Glu Gln Leu Phe Lys Ser SerLys Leu Tyr Tyr 195 200 205 gtt atg aag ctg ctc acg aat gtt gct att tttgct gcg agc att gca 672 Val Met Lys Leu Leu Thr Asn Val Ala Ile Phe AlaAla Ser Ile Ala 210 215 220 ata ata tgt tgg agc aag act att tca gcg gttttg gct tca gct tgt 720 Ile Ile Cys Trp Ser Lys Thr Ile Ser Ala Val LeuAla Ser Ala Cys 225 230 235 240 atg atg gct ctg tgt ttc caa cag tgc ggatgg cta tcc cat gat ttt 768 Met Met Ala Leu Cys Phe Gln Gln Cys Gly TrpLeu Ser His Asp Phe 245 250 255 ctc cac aat cag gtg ttt gag aca cgc tggctt aat gaa gtt gtc ggg 816 Leu His Asn Gln Val Phe Glu Thr Arg Trp LeuAsn Glu Val Val Gly 260 265 270 tat gtg atc ggc aac gcc gtt ctg ggg tttagt aca ggg tgg tgg aag 864 Tyr Val Ile Gly Asn Ala Val Leu Gly Phe SerThr Gly Trp Trp Lys 275 280 285 gag aag cat aac ctt cat cat gct gct ccaaat gaa tgc gat cag act 912 Glu Lys His Asn Leu His His Ala Ala Pro AsnGlu Cys Asp Gln Thr 290 295 300 tac caa cca att gat gaa gat att gat actctc ccc ctc att gcc tgg 960 Tyr Gln Pro Ile Asp Glu Asp Ile Asp Thr LeuPro Leu Ile Ala Trp 305 310 315 320 agc aag gac ata ctg gcc aca gtt gagaat aag aca ttc ttg cga atc 1008 Ser Lys Asp Ile Leu Ala Thr Val Glu AsnLys Thr Phe Leu Arg Ile 325 330 335 ctc caa tac cag cat ctg ttc ttc atgggt ctg tta ttt ttc gcc cgt 1056 Leu Gln Tyr Gln His Leu Phe Phe Met GlyLeu Leu Phe Phe Ala Arg 340 345 350 ggt agt tgg ctc ttt tgg agc tgg agatat acc tct aca gca gtg ctc 1104 Gly Ser Trp Leu Phe Trp Ser Trp Arg TyrThr Ser Thr Ala Val Leu 355 360 365 tca cct gtc gac agg ttg ttg gag aaggga act gtt ctg ttt cac tac 1152 Ser Pro Val Asp Arg Leu Leu Glu Lys GlyThr Val Leu Phe His Tyr 370 375 380 ttt tgg ttc gtc ggg aca gcg tgc tatctt ctc cct ggt tgg aag cca 1200 Phe Trp Phe Val Gly Thr Ala Cys Tyr LeuLeu Pro Gly Trp Lys Pro 385 390 395 400 tta gta tgg atg gcg gtg act gagctc atg tcc ggc atg ctg ctg ggc 1248 Leu Val Trp Met Ala Val Thr Glu LeuMet Ser Gly Met Leu Leu Gly 405 410 415 ttt gta ttt gta ctt agc cac aatggg atg gag gtt tat aat tcg tct 1296 Phe Val Phe Val Leu Ser His Asn GlyMet Glu Val Tyr Asn Ser Ser 420 425 430 aaa gaa ttc gtg agt gca cag atcgta tcc aca cgg gat atc aaa gga 1344 Lys Glu Phe Val Ser Ala Gln Ile ValSer Thr Arg Asp Ile Lys Gly 435 440 445 aac ata ttc aac gac tgg ttc actggt ggc ctt aac agg caa ata gag 1392 Asn Ile Phe Asn Asp Trp Phe Thr GlyGly Leu Asn Arg Gln Ile Glu 450 455 460 cat cat ctt ttc cca aca atg cccagg cat aat tta aac aaa ata gca 1440 His His Leu Phe Pro Thr Met Pro ArgHis Asn Leu Asn Lys Ile Ala 465 470 475 480 cct aga gtg gag gtg ttc tgtaag aaa cac ggt ctg gtg tac gaa gac 1488 Pro Arg Val Glu Val Phe Cys LysLys His Gly Leu Val Tyr Glu Asp 485 490 495 gta tct att gct acc ggc acttgc aag gtt ttg aaa gca ttg aag gaa 1536 Val Ser Ile Ala Thr Gly Thr CysLys Val Leu Lys Ala Leu Lys Glu 500 505 510 gtc gcg gag gct gcg gca gagcag cat gct acc acc agt taa 1578 Val Ala Glu Ala Ala Ala Glu Gln His AlaThr Thr Ser 515 520 525 8 525 PRT Physcomitrella patens 8 Met Val PheAla Gly Gly Gly Leu Gln Gln Gly Ser Leu Glu Glu Asn 1 5 10 15 Ile AspVal Glu His Ile Ala Ser Met Ser Leu Phe Ser Asp Phe Phe 20 25 30 Ser TyrVal Ser Ser Thr Val Gly Ser Trp Ser Val His Ser Ile Gln 35 40 45 Pro LeuLys Arg Leu Thr Ser Lys Lys Arg Val Ser Glu Ser Ala Ala 50 55 60 Val GlnCys Ile Ser Ala Glu Val Gln Arg Asn Ser Ser Thr Gln Gly 65 70 75 80 ThrAla Glu Ala Leu Ala Glu Ser Val Val Lys Pro Thr Arg Arg Arg 85 90 95 SerSer Gln Trp Lys Lys Ser Thr His Pro Leu Ser Glu Val Ala Val 100 105 110His Asn Lys Pro Ser Asp Cys Trp Ile Val Val Lys Asn Lys Val Tyr 115 120125 Asp Val Ser Asn Phe Ala Asp Glu His Pro Gly Gly Ser Val Ile Ser 130135 140 Thr Tyr Phe Gly Arg Asp Gly Thr Asp Val Phe Ser Ser Phe His Ala145 150 155 160 Ala Ser Thr Trp Lys Ile Leu Gln Asp Phe Tyr Ile Gly AspVal Glu 165 170 175 Arg Val Glu Pro Thr Pro Glu Leu Leu Lys Asp Phe ArgGlu Met Arg 180 185 190 Ala Leu Phe Leu Arg Glu Gln Leu Phe Lys Ser SerLys Leu Tyr Tyr 195 200 205 Val Met Lys Leu Leu Thr Asn Val Ala Ile PheAla Ala Ser Ile Ala 210 215 220 Ile Ile Cys Trp Ser Lys Thr Ile Ser AlaVal Leu Ala Ser Ala Cys 225 230 235 240 Met Met Ala Leu Cys Phe Gln GlnCys Gly Trp Leu Ser His Asp Phe 245 250 255 Leu His Asn Gln Val Phe GluThr Arg Trp Leu Asn Glu Val Val Gly 260 265 270 Tyr Val Ile Gly Asn AlaVal Leu Gly Phe Ser Thr Gly Trp Trp Lys 275 280 285 Glu Lys His Asn LeuHis His Ala Ala Pro Asn Glu Cys Asp Gln Thr 290 295 300 Tyr Gln Pro IleAsp Glu Asp Ile Asp Thr Leu Pro Leu Ile Ala Trp 305 310 315 320 Ser LysAsp Ile Leu Ala Thr Val Glu Asn Lys Thr Phe Leu Arg Ile 325 330 335 LeuGln Tyr Gln His Leu Phe Phe Met Gly Leu Leu Phe Phe Ala Arg 340 345 350Gly Ser Trp Leu Phe Trp Ser Trp Arg Tyr Thr Ser Thr Ala Val Leu 355 360365 Ser Pro Val Asp Arg Leu Leu Glu Lys Gly Thr Val Leu Phe His Tyr 370375 380 Phe Trp Phe Val Gly Thr Ala Cys Tyr Leu Leu Pro Gly Trp Lys Pro385 390 395 400 Leu Val Trp Met Ala Val Thr Glu Leu Met Ser Gly Met LeuLeu Gly 405 410 415 Phe Val Phe Val Leu Ser His Asn Gly Met Glu Val TyrAsn Ser Ser 420 425 430 Lys Glu Phe Val Ser Ala Gln Ile Val Ser Thr ArgAsp Ile Lys Gly 435 440 445 Asn Ile Phe Asn Asp Trp Phe Thr Gly Gly LeuAsn Arg Gln Ile Glu 450 455 460 His His Leu Phe Pro Thr Met Pro Arg HisAsn Leu Asn Lys Ile Ala 465 470 475 480 Pro Arg Val Glu Val Phe Cys LysLys His Gly Leu Val Tyr Glu Asp 485 490 495 Val Ser Ile Ala Thr Gly ThrCys Lys Val Leu Lys Ala Leu Lys Glu 500 505 510 Val Ala Glu Ala Ala AlaGlu Gln His Ala Thr Thr Ser 515 520 525 9 873 DNA Physcomitrella patensCDS (1)..(873) 9 atg gag gtc gtg gag aga ttc tac ggt gag ttg gat ggg aaggtc tcg 48 Met Glu Val Val Glu Arg Phe Tyr Gly Glu Leu Asp Gly Lys ValSer 1 5 10 15 cag ggc gtg aat gca ttg ctg ggt agt ttt ggg gtg gag ttgacg gat 96 Gln Gly Val Asn Ala Leu Leu Gly Ser Phe Gly Val Glu Leu ThrAsp 20 25 30 acg ccc act acc aaa ggc ttg ccc ctc gtt gac agt ccc aca cccatc 144 Thr Pro Thr Thr Lys Gly Leu Pro Leu Val Asp Ser Pro Thr Pro Ile35 40 45 gtc ctc ggt gtt tct gta tac ttg act att gtc att gga ggg ctt ttg192 Val Leu Gly Val Ser Val Tyr Leu Thr Ile Val Ile Gly Gly Leu Leu 5055 60 tgg ata aag gcc agg gat ctg aaa ccg cgc gcc tcg gag cca ttt ttg240 Trp Ile Lys Ala Arg Asp Leu Lys Pro Arg Ala Ser Glu Pro Phe Leu 6570 75 80 ctc caa gct ttg gtg ctt gtg cac aac ctg ttc tgt ttt gcg ctc agt288 Leu Gln Ala Leu Val Leu Val His Asn Leu Phe Cys Phe Ala Leu Ser 8590 95 ctg tat atg tgc gtg ggc atc gct tat cag gct att acc tgg cgg tac336 Leu Tyr Met Cys Val Gly Ile Ala Tyr Gln Ala Ile Thr Trp Arg Tyr 100105 110 tct ctc tgg ggc aat gca tac aat cct aaa cat aaa gag atg gcg att384 Ser Leu Trp Gly Asn Ala Tyr Asn Pro Lys His Lys Glu Met Ala Ile 115120 125 ctg gta tac ttg ttc tac atg tct aag tac gtg gaa ttc atg gat acc432 Leu Val Tyr Leu Phe Tyr Met Ser Lys Tyr Val Glu Phe Met Asp Thr 130135 140 gtt atc atg ata ctg aag cgc agc acc agg caa ata agc ttc ctc cac480 Val Ile Met Ile Leu Lys Arg Ser Thr Arg Gln Ile Ser Phe Leu His 145150 155 160 gtt tat cat cat tct tca att tcc ctc att tgg tgg gct att gctcat 528 Val Tyr His His Ser Ser Ile Ser Leu Ile Trp Trp Ala Ile Ala His165 170 175 cac gct cct ggc ggt gaa gca tat tgg tct gcg gct ctg aac tcagga 576 His Ala Pro Gly Gly Glu Ala Tyr Trp Ser Ala Ala Leu Asn Ser Gly180 185 190 gtg cat gtt ctc atg tat gcg tat tac ttc ttg gct gcc tgc cttcga 624 Val His Val Leu Met Tyr Ala Tyr Tyr Phe Leu Ala Ala Cys Leu Arg195 200 205 agt agc cca aag tta aaa aat aag tac ctt ttt tgg ggc agg tacttg 672 Ser Ser Pro Lys Leu Lys Asn Lys Tyr Leu Phe Trp Gly Arg Tyr Leu210 215 220 aca caa ttc caa atg ttc cag ttt atg ctg aac tta gtg cag gcttac 720 Thr Gln Phe Gln Met Phe Gln Phe Met Leu Asn Leu Val Gln Ala Tyr225 230 235 240 tac gac atg aaa acg aat gcg cca tat cca caa tgg ctg atcaag att 768 Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro Gln Trp Leu Ile LysIle 245 250 255 ttg ttc tac tac atg atc tcg ttg ctg ttt ctt ttc ggc aatttt tac 816 Leu Phe Tyr Tyr Met Ile Ser Leu Leu Phe Leu Phe Gly Asn PheTyr 260 265 270 gta caa aaa tac atc aaa ccc tct gac gga aag caa aag ggagct aaa 864 Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly Lys Gln Lys Gly AlaLys 275 280 285 act gag tga 873 Thr Glu 290 10 290 PRT Physcomitrellapatens 10 Met Glu Val Val Glu Arg Phe Tyr Gly Glu Leu Asp Gly Lys ValSer 1 5 10 15 Gln Gly Val Asn Ala Leu Leu Gly Ser Phe Gly Val Glu LeuThr Asp 20 25 30 Thr Pro Thr Thr Lys Gly Leu Pro Leu Val Asp Ser Pro ThrPro Ile 35 40 45 Val Leu Gly Val Ser Val Tyr Leu Thr Ile Val Ile Gly GlyLeu Leu 50 55 60 Trp Ile Lys Ala Arg Asp Leu Lys Pro Arg Ala Ser Glu ProPhe Leu 65 70 75 80 Leu Gln Ala Leu Val Leu Val His Asn Leu Phe Cys PheAla Leu Ser 85 90 95 Leu Tyr Met Cys Val Gly Ile Ala Tyr Gln Ala Ile ThrTrp Arg Tyr 100 105 110 Ser Leu Trp Gly Asn Ala Tyr Asn Pro Lys His LysGlu Met Ala Ile 115 120 125 Leu Val Tyr Leu Phe Tyr Met Ser Lys Tyr ValGlu Phe Met Asp Thr 130 135 140 Val Ile Met Ile Leu Lys Arg Ser Thr ArgGln Ile Ser Phe Leu His 145 150 155 160 Val Tyr His His Ser Ser Ile SerLeu Ile Trp Trp Ala Ile Ala His 165 170 175 His Ala Pro Gly Gly Glu AlaTyr Trp Ser Ala Ala Leu Asn Ser Gly 180 185 190 Val His Val Leu Met TyrAla Tyr Tyr Phe Leu Ala Ala Cys Leu Arg 195 200 205 Ser Ser Pro Lys LeuLys Asn Lys Tyr Leu Phe Trp Gly Arg Tyr Leu 210 215 220 Thr Gln Phe GlnMet Phe Gln Phe Met Leu Asn Leu Val Gln Ala Tyr 225 230 235 240 Tyr AspMet Lys Thr Asn Ala Pro Tyr Pro Gln Trp Leu Ile Lys Ile 245 250 255 LeuPhe Tyr Tyr Met Ile Ser Leu Leu Phe Leu Phe Gly Asn Phe Tyr 260 265 270Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly Lys Gln Lys Gly Ala Lys 275 280285 Thr Glu 290 11 1526 DNA Phaeodactylum tricornutum CDS (92)..(1402)11 gcttccgtta gcgtcccata gtttgttaca cttggctgtg aaacgaatac gttcttggtc 60tacttactac aacgaagcaa ccaccagcag c atg ggt aag gga ggt caa cga 112 MetGly Lys Gly Gly Gln Arg 1 5 gct gta gct ccc aag agt gcc acc agc tct actggc agt gct acc ctt 160 Ala Val Ala Pro Lys Ser Ala Thr Ser Ser Thr GlySer Ala Thr Leu 10 15 20 agc caa agc aag gaa cag gta tgg act tcg tcg tacaac cct ctg gcg 208 Ser Gln Ser Lys Glu Gln Val Trp Thr Ser Ser Tyr AsnPro Leu Ala 25 30 35 aag gat tcc ccg gag ctg cca acc aaa ggc caa atc aaggcc gtc att 256 Lys Asp Ser Pro Glu Leu Pro Thr Lys Gly Gln Ile Lys AlaVal Ile 40 45 50 55 ccg aag gaa tgt ttc caa cgc tca gcc ttt tgg tct accttc tac ctg 304 Pro Lys Glu Cys Phe Gln Arg Ser Ala Phe Trp Ser Thr PheTyr Leu 60 65 70 atg cgc gat ctc gcc atg gct gcc gcc ttt tgc tac gga acctca cag 352 Met Arg Asp Leu Ala Met Ala Ala Ala Phe Cys Tyr Gly Thr SerGln 75 80 85 gtc ctc tcc acc gac ctt ccc caa gac gcc acg ctc att ctg ccctgg 400 Val Leu Ser Thr Asp Leu Pro Gln Asp Ala Thr Leu Ile Leu Pro Trp90 95 100 gct ctc ggc tgg ggc gtc tac gcc ttt tgg atg gga acc att ctcacc 448 Ala Leu Gly Trp Gly Val Tyr Ala Phe Trp Met Gly Thr Ile Leu Thr105 110 115 ggg cct tgg gta gtt gcg cac gaa tgt gga cac ggc gct tac tccgac 496 Gly Pro Trp Val Val Ala His Glu Cys Gly His Gly Ala Tyr Ser Asp120 125 130 135 tcc cag acg ttc aat gac gtg gtc ggc ttt atc gtc cac caagct ttg 544 Ser Gln Thr Phe Asn Asp Val Val Gly Phe Ile Val His Gln AlaLeu 140 145 150 ctc gtc ccc tac ttt gcc tgg cag tac acc cac gcg aaa caccac cgt 592 Leu Val Pro Tyr Phe Ala Trp Gln Tyr Thr His Ala Lys His HisArg 155 160 165 cga acc aac cat ctg gtg gac ggc gag tcc cac gtc cct tctacc gcc 640 Arg Thr Asn His Leu Val Asp Gly Glu Ser His Val Pro Ser ThrAla 170 175 180 aag gat aac ggc ctc ggg ccg cac aac gag cga aac tcc ttctac gcc 688 Lys Asp Asn Gly Leu Gly Pro His Asn Glu Arg Asn Ser Phe TyrAla 185 190 195 gcg tgg cac gag gcc atg gga gac ggc gcc ttt gcc gtc tttcaa gtc 736 Ala Trp His Glu Ala Met Gly Asp Gly Ala Phe Ala Val Phe GlnVal 200 205 210 215 tgg tcg cac ttg ttc gtc ggc tgg cct ctc tac ttg gccggt ctg gcc 784 Trp Ser His Leu Phe Val Gly Trp Pro Leu Tyr Leu Ala GlyLeu Ala 220 225 230 agt acc gga aag ctt gcg cac gaa ggt tgg tgg ctg gaagaa cgg aac 832 Ser Thr Gly Lys Leu Ala His Glu Gly Trp Trp Leu Glu GluArg Asn 235 240 245 gcg att gcg gat cac ttt cga ccc agc tct ccc atg ttcccc gcc aag 880 Ala Ile Ala Asp His Phe Arg Pro Ser Ser Pro Met Phe ProAla Lys 250 255 260 atc cgt gcc aag att gcc ctt tcc agc gcg acg gaa ctcgcc gtg ctc 928 Ile Arg Ala Lys Ile Ala Leu Ser Ser Ala Thr Glu Leu AlaVal Leu 265 270 275 gct gga ctc ttg tat gtc ggt aca cag gtc gga cac cttccc gtc ctg 976 Ala Gly Leu Leu Tyr Val Gly Thr Gln Val Gly His Leu ProVal Leu 280 285 290 295 ctg tgg tac tgg gga ccg tac acc ttt gtc aac gcttgg ctt gta ctc 1024 Leu Trp Tyr Trp Gly Pro Tyr Thr Phe Val Asn Ala TrpLeu Val Leu 300 305 310 tac acg tgg ctg cag cat acg gac ccg tcc atc ccgcac tac ggt gaa 1072 Tyr Thr Trp Leu Gln His Thr Asp Pro Ser Ile Pro HisTyr Gly Glu 315 320 325 ggc gag tgg acc tgg gtc aag ggc gcg ctc tct accatt gat cga gac 1120 Gly Glu Trp Thr Trp Val Lys Gly Ala Leu Ser Thr IleAsp Arg Asp 330 335 340 tac ggc atc ttc gat ttc ttt cac cac acc atc ggttcc acg cac gtg 1168 Tyr Gly Ile Phe Asp Phe Phe His His Thr Ile Gly SerThr His Val 345 350 355 gta cac cat ttg ttc cac gaa atg ccc tgg tac aatgcc ggc att gcc 1216 Val His His Leu Phe His Glu Met Pro Trp Tyr Asn AlaGly Ile Ala 360 365 370 375 acg caa aag gtc aag gaa ttt ttg gaa ccc cagggc ttg tac aat tac 1264 Thr Gln Lys Val Lys Glu Phe Leu Glu Pro Gln GlyLeu Tyr Asn Tyr 380 385 390 gat ccg acc ccc tgg tac aag gcc atg tgg cgcatt gcc cgg acc tgt 1312 Asp Pro Thr Pro Trp Tyr Lys Ala Met Trp Arg IleAla Arg Thr Cys 395 400 405 cac tat gtg gag tca aac gag ggt gtg cag tatttc aag agt atg gaa 1360 His Tyr Val Glu Ser Asn Glu Gly Val Gln Tyr PheLys Ser Met Glu 410 415 420 aac gtg ccg ctg act aag gat gtg cga aac aaagcc gca tga 1402 Asn Val Pro Leu Thr Lys Asp Val Arg Asn Lys Ala Ala 425430 435 gaaaaagtgc caccgacgca taattttaca atcctaccaa caagaccaacattatatggt 1462 tttcgcttaa aagatagttt tttctaccat ctgtgtagtc ggcacaaaaaaaaaaaaaaa 1522 aaaa 1526 12 436 PRT Phaeodactylum tricornutum 12 MetGly Lys Gly Gly Gln Arg Ala Val Ala Pro Lys Ser Ala Thr Ser 1 5 10 15Ser Thr Gly Ser Ala Thr Leu Ser Gln Ser Lys Glu Gln Val Trp Thr 20 25 30Ser Ser Tyr Asn Pro Leu Ala Lys Asp Ser Pro Glu Leu Pro Thr Lys 35 40 45Gly Gln Ile Lys Ala Val Ile Pro Lys Glu Cys Phe Gln Arg Ser Ala 50 55 60Phe Trp Ser Thr Phe Tyr Leu Met Arg Asp Leu Ala Met Ala Ala Ala 65 70 7580 Phe Cys Tyr Gly Thr Ser Gln Val Leu Ser Thr Asp Leu Pro Gln Asp 85 9095 Ala Thr Leu Ile Leu Pro Trp Ala Leu Gly Trp Gly Val Tyr Ala Phe 100105 110 Trp Met Gly Thr Ile Leu Thr Gly Pro Trp Val Val Ala His Glu Cys115 120 125 Gly His Gly Ala Tyr Ser Asp Ser Gln Thr Phe Asn Asp Val ValGly 130 135 140 Phe Ile Val His Gln Ala Leu Leu Val Pro Tyr Phe Ala TrpGln Tyr 145 150 155 160 Thr His Ala Lys His His Arg Arg Thr Asn His LeuVal Asp Gly Glu 165 170 175 Ser His Val Pro Ser Thr Ala Lys Asp Asn GlyLeu Gly Pro His Asn 180 185 190 Glu Arg Asn Ser Phe Tyr Ala Ala Trp HisGlu Ala Met Gly Asp Gly 195 200 205 Ala Phe Ala Val Phe Gln Val Trp SerHis Leu Phe Val Gly Trp Pro 210 215 220 Leu Tyr Leu Ala Gly Leu Ala SerThr Gly Lys Leu Ala His Glu Gly 225 230 235 240 Trp Trp Leu Glu Glu ArgAsn Ala Ile Ala Asp His Phe Arg Pro Ser 245 250 255 Ser Pro Met Phe ProAla Lys Ile Arg Ala Lys Ile Ala Leu Ser Ser 260 265 270 Ala Thr Glu LeuAla Val Leu Ala Gly Leu Leu Tyr Val Gly Thr Gln 275 280 285 Val Gly HisLeu Pro Val Leu Leu Trp Tyr Trp Gly Pro Tyr Thr Phe 290 295 300 Val AsnAla Trp Leu Val Leu Tyr Thr Trp Leu Gln His Thr Asp Pro 305 310 315 320Ser Ile Pro His Tyr Gly Glu Gly Glu Trp Thr Trp Val Lys Gly Ala 325 330335 Leu Ser Thr Ile Asp Arg Asp Tyr Gly Ile Phe Asp Phe Phe His His 340345 350 Thr Ile Gly Ser Thr His Val Val His His Leu Phe His Glu Met Pro355 360 365 Trp Tyr Asn Ala Gly Ile Ala Thr Gln Lys Val Lys Glu Phe LeuGlu 370 375 380 Pro Gln Gly Leu Tyr Asn Tyr Asp Pro Thr Pro Trp Tyr LysAla Met 385 390 395 400 Trp Arg Ile Ala Arg Thr Cys His Tyr Val Glu SerAsn Glu Gly Val 405 410 415 Gln Tyr Phe Lys Ser Met Glu Asn Val Pro LeuThr Lys Asp Val Arg 420 425 430 Asn Lys Ala Ala 435 13 3598 DNA UnknownSequence represents a plant promoter-terminator expression cassette invector pUC19 13 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccggagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcgtcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgtactgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgcatcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcctcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggtaacgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cggcgcgccgagctcctcga 420 gcaaatttac acattgccac taaacgtcta aacccttgta atttgtttttgttttactat 480 gtgtgttatg tatttgattt gcgataaatt tttatatttg gtactaaatttataacacct 540 tttatgctaa cgtttgccaa cacttagcaa tttgcaagtt gattaattgattctaaatta 600 tttttgtctt ctaaatacat atactaatca actggaaatg taaatatttgctaatatttc 660 tactatagga gaattaaagt gagtgaatat ggtaccacaa ggtttggagatttaattgtt 720 gcaatgctgc atggatggca tatacaccaa acattcaata attcttgaggataataatgg 780 taccacacaa gatttgaggt gcatgaacgt cacgtggaca aaaggtttagtaatttttca 840 agacaacaat gttaccacac acaagttttg aggtgcatgc atggatgccctgtggaaagt 900 ttaaaaatat tttggaaatg atttgcatgg aagccatgtg taaaaccatgacatccactt 960 ggaggatgca ataatgaaga aaactacaaa tttacatgca actagttatgcatgtagtct 1020 atataatgag gattttgcaa tactttcatt catacacact cactaagttttacacgatta 1080 taatttcttc atagccagcc caccgcggtg ggcggccgcc tgcagtctagaaggcctcct 1140 gctttaatga gatatgcgag acgcctatga tcgcatgata tttgctttcaattctgttgt 1200 gcacgttgta aaaaacctga gcatgtgtag ctcagatcct taccgccggtttcggttcat 1260 tctaatgaat atatcacccg ttactatcgt atttttatga ataatattctccgttcaatt 1320 tactgattgt ccgtcgacga attcgagctc ggcgcgccaa gcttggcgtaatcatggtca 1380 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacatacgagccgga 1440 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacattaattgcgttg 1500 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcattaatgaatcggc 1560 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctcgctcactgac 1620 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaaggcggtaata 1680 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaaaggccagcaa 1740 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggctccgcccccct 1800 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgacaggactataa 1860 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttccgaccctgccg 1920 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttctcatagctca 1980 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctgtgtgcacgaa 2040 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttgagtccaacccg 2100 gtaagacacg acttatcgcc actggcagca gccactggta acaggattagcagagcgagg 2160 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggctacactagaagg 2220 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaagagttggtagc 2280 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttgcaagcagcag 2340 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctacggggtctgac 2400 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatcaaaaaggatc 2460 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaagtatatatgag 2520 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctcagcgatctgt 2580 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactacgatacgggag 2640 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctcaccggctcca 2700 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtggtcctgcaact 2760 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaagtagttcgcca 2820 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtcacgctcgtcg 2880 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttacatgatccccc 2940 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcagaagtaagttg 3000 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttactgtcatgcca 3060 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctgagaatagtgt 3120 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgcgccacatagc 3180 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaactctcaaggatc 3240 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactgatcttcagca 3300 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaatgccgcaaaa 3360 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttttcaatattat 3420 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatgtatttagaaa 3480 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctgacgtctaagaa 3540 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggccctttcgtc 3598 14 3590 DNA Unknown Sequence represents a plantpromoter-terminator expression cassette in vector pUC19 14 tcgcgcgtttcggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtctgtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtgtcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcggtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccattcaggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagctggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtcacgacgttgt aaaacgacgg ccagtgaatt cggcgcgccg agctcctcga 420 gcaaatttacacattgccac taaacgtcta aacccttgta atttgttttt gttttactat 480 gtgtgttatgtatttgattt gcgataaatt tttatatttg gtactaaatt tataacacct 540 tttatgctaacgtttgccaa cacttagcaa tttgcaagtt gattaattga ttctaaatta 600 tttttgtcttctaaatacat atactaatca actggaaatg taaatatttg ctaatatttc 660 tactataggagaattaaagt gagtgaatat ggtaccacaa ggtttggaga tttaattgtt 720 gcaatgctgcatggatggca tatacaccaa acattcaata attcttgagg ataataatgg 780 taccacacaagatttgaggt gcatgaacgt cacgtggaca aaaggtttag taatttttca 840 agacaacaatgttaccacac acaagttttg aggtgcatgc atggatgccc tgtggaaagt 900 ttaaaaatattttggaaatg atttgcatgg aagccatgtg taaaaccatg acatccactt 960 ggaggatgcaataatgaaga aaactacaaa tttacatgca actagttatg catgtagtct 1020 atataatgaggattttgcaa tactttcatt catacacact cactaagttt tacacgatta 1080 taatttcttcatagccagcg gatccgatat cgggcccgct agcgttaacc ctgctttaat 1140 gagatatgcgagacgcctat gatcgcatga tatttgcttt caattctgtt gtgcacgttg 1200 taaaaaacctgagcatgtgt agctcagatc cttaccgccg gtttcggttc attctaatga 1260 atatatcacccgttactatc gtatttttat gaataatatt ctccgttcaa tttactgatt 1320 gtccgtcgacgaattcgagc tcggcgcgcc aagcttggcg taatcatggt catagctgtt 1380 tcctgtgtgaaattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 1440 gtgtaaagcctggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 1500 gcccgctttccagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 1560 ggggagaggcggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 1620 ctcggtcgttcggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 1680 cacagaatcaggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 1740 gaaccgtaaaaaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 1800 tcacaaaaatcgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 1860 ggcgtttccccctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 1920 atacctgtccgcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 1980 gtatctcagttcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 2040 tcagcccgaccgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 2100 cgacttatcgccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 2160 cggtgctacagagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 2220 tggtatctgcgctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 2280 cggcaaacaaaccaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 2340 cagaaaaaaaggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 2400 gaacgaaaactcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 2460 gatccttttaaattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 2520 gtctgacagttaccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 2580 ttcatccatagttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 2640 atctggccccagtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 2700 agcaataaaccagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 2760 ctccatccagtctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 2820 tttgcgcaacgttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 2880 ggcttcattcagctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 2940 caaaaaagcggttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 3000 gttatcactcatggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 3060 atgcttttctgtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 3120 accgagttgctcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 3180 aaaagtgctcatcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 3240 gttgagatccagttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 3300 tttcaccagcgtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 3360 aagggcgacacggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 3420 ttatcagggttattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 3480 aataggggttccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat 3540 tatcatgacattaacctata aaaataggcg tatcacgagg ccctttcgtc 3590 15 3584 DNA UnknownSequence represents a plant promoter-terminator expression cassette invector pUC19 15 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccggagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcgtcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgtactgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgcatcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcctcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggtaacgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cggcgcgccgagctcctcga 420 gcaaatttac acattgccac taaacgtcta aacccttgta atttgtttttgttttactat 480 gtgtgttatg tatttgattt gcgataaatt tttatatttg gtactaaatttataacacct 540 tttatgctaa cgtttgccaa cacttagcaa tttgcaagtt gattaattgattctaaatta 600 tttttgtctt ctaaatacat atactaatca actggaaatg taaatatttgctaatatttc 660 tactatagga gaattaaagt gagtgaatat ggtaccacaa ggtttggagatttaattgtt 720 gcaatgctgc atggatggca tatacaccaa acattcaata attcttgaggataataatgg 780 taccacacaa gatttgaggt gcatgaacgt cacgtggaca aaaggtttagtaatttttca 840 agacaacaat gttaccacac acaagttttg aggtgcatgc atggatgccctgtggaaagt 900 ttaaaaatat tttggaaatg atttgcatgg aagccatgtg taaaaccatgacatccactt 960 ggaggatgca ataatgaaga aaactacaaa tttacatgca actagttatgcatgtagtct 1020 atataatgag gattttgcaa tactttcatt catacacact cactaagttttacacgatta 1080 taatttcttc atagccagca gatctgccgg catcgatccc gggccatggcctgctttaat 1140 gagatatgcg agacgcctat gatcgcatga tatttgcttt caattctgttgtgcacgttg 1200 taaaaaacct gagcatgtgt agctcagatc cttaccgccg gtttcggttcattctaatga 1260 atatatcacc cgttactatc gtatttttat gaataatatt ctccgttcaatttactgatt 1320 gtccgtcgac gagctcggcg cgccaagctt ggcgtaatca tggtcatagctgtttcctgt 1380 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagcataaagtgtaa 1440 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgctcactgcccgc 1500 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaacgcgcggggag 1560 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgctgcgctcggt 1620 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggttatccacaga 1680 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaaggccaggaaccg 1740 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacgagcatcacaa 1800 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagataccaggcgtt 1860 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgcttaccggatacct 1920 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgctgtaggtatct 1980 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaaccccccgttcagcc 2040 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaagacacgactt 2100 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatgtaggcggtgc 2160 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacagtatttggtat 2220 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctcttgatccggcaa 2280 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagattacgcgcagaaa 2340 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctcagtggaacga 2400 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttcacctagatcct 2460 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaacttggtctga 2520 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctatttcgttcatc 2580 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggcttaccatctgg 2640 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatttatcagcaat 2700 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttatccgcctccat 2760 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagttaatagtttgcg 2820 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttggtatggcttc 2880 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgttgtgcaaaaa 2940 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccgcagtgttatc 3000 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccgtaagatgctt 3060 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgcggcgaccgag 3120 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaactttaaaagt 3180 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttaccgctgttgag 3240 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatcttttactttcac 3300 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagggaataagggc 3360 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaagcatttatca 3420 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaataaacaaatagg 3480 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaaccattattatcat 3540 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc 3584 164507 DNA Unknown Sequence represents a plant promoter-terminatorexpression cassette in vector pUC19 16 tcgcgcgttt cggtgatgac ggtgaaaacctctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagcagacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatgcggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagatgcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttgggaagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctgcaaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacggccagtgaatt cggcgcgccg agctcctcga 420 gcaaatttac acattgccac taaacgtctaaacccttgta atttgttttt gttttactat 480 gtgtgttatg tatttgattt gcgataaatttttatatttg gtactaaatt tataacacct 540 tttatgctaa cgtttgccaa cacttagcaatttgcaagtt gattaattga ttctaaatta 600 tttttgtctt ctaaatacat atactaatcaactggaaatg taaatatttg ctaatatttc 660 tactatagga gaattaaagt gagtgaatatggtaccacaa ggtttggaga tttaattgtt 720 gcaatgctgc atggatggca tatacaccaaacattcaata attcttgagg ataataatgg 780 taccacacaa gatttgaggt gcatgaacgtcacgtggaca aaaggtttag taatttttca 840 agacaacaat gttaccacac acaagttttgaggtgcatgc atggatgccc tgtggaaagt 900 ttaaaaatat tttggaaatg atttgcatggaagccatgtg taaaaccatg acatccactt 960 ggaggatgca ataatgaaga aaactacaaatttacatgca actagttatg catgtagtct 1020 atataatgag gattttgcaa tactttcattcatacacact cactaagttt tacacgatta 1080 taatttcttc atagccagcc caccgcggtgggcggccgcc tgcagtctag aaggcctcct 1140 gctttaatga gatatgcgag acgcctatgatcgcatgata tttgctttca attctgttgt 1200 gcacgttgta aaaaacctga gcatgtgtagctcagatcct taccgccggt ttcggttcat 1260 tctaatgaat atatcacccg ttactatcgtatttttatga ataatattct ccgttcaatt 1320 tactgattgt ccgtcgagca aatttacacattgccactaa acgtctaaac ccttgtaatt 1380 tgtttttgtt ttactatgtg tgttatgtatttgatttgcg ataaattttt atatttggta 1440 ctaaatttat aacacctttt atgctaacgtttgccaacac ttagcaattt gcaagttgat 1500 taattgattc taaattattt ttgtcttctaaatacatata ctaatcaact ggaaatgtaa 1560 atatttgcta atatttctac tataggagaattaaagtgag tgaatatggt accacaaggt 1620 ttggagattt aattgttgca atgctgcatggatggcatat acaccaaaca ttcaataatt 1680 cttgaggata ataatggtac cacacaagatttgaggtgca tgaacgtcac gtggacaaaa 1740 ggtttagtaa tttttcaaga caacaatgttaccacacaca agttttgagg tgcatgcatg 1800 gatgccctgt ggaaagttta aaaatattttggaaatgatt tgcatggaag ccatgtgtaa 1860 aaccatgaca tccacttgga ggatgcaataatgaagaaaa ctacaaattt acatgcaact 1920 agttatgcat gtagtctata taatgaggattttgcaatac tttcattcat acacactcac 1980 taagttttac acgattataa tttcttcatagccagcggat ccgatatcgg gcccgctagc 2040 gttaaccctg ctttaatgag atatgcgagacgcctatgat cgcatgatat ttgctttcaa 2100 ttctgttgtg cacgttgtaa aaaacctgagcatgtgtagc tcagatcctt accgccggtt 2160 tcggttcatt ctaatgaata tatcacccgttactatcgta tttttatgaa taatattctc 2220 cgttcaattt actgattgtc cgtcgacgaattcgagctcg gcgcgccaag cttggcgtaa 2280 tcatggtcat agctgtttcc tgtgtgaaattgttatccgc tcacaattcc acacaacata 2340 cgagccggaa gcataaagtg taaagcctggggtgcctaat gagtgagcta actcacatta 2400 attgcgttgc gctcactgcc cgctttccagtcgggaaacc tgtcgtgcca gctgcattaa 2460 tgaatcggcc aacgcgcggg gagaggcggtttgcgtattg ggcgctcttc cgcttcctcg 2520 ctcactgact cgctgcgctc ggtcgttcggctgcggcgag cggtatcagc tcactcaaag 2580 gcggtaatac ggttatccac agaatcaggggataacgcag gaaagaacat gtgagcaaaa 2640 ggccagcaaa aggccaggaa ccgtaaaaaggccgcgttgc tggcgttttt ccataggctc 2700 cgcccccctg acgagcatca caaaaatcgacgctcaagtc agaggtggcg aaacccgaca 2760 ggactataaa gataccaggc gtttccccctggaagctccc tcgtgcgctc tcctgttccg 2820 accctgccgc ttaccggata cctgtccgcctttctccctt cgggaagcgt ggcgctttct 2880 catagctcac gctgtaggta tctcagttcggtgtaggtcg ttcgctccaa gctgggctgt 2940 gtgcacgaac cccccgttca gcccgaccgctgcgccttat ccggtaacta tcgtcttgag 3000 tccaacccgg taagacacga cttatcgccactggcagcag ccactggtaa caggattagc 3060 agagcgaggt atgtaggcgg tgctacagagttcttgaagt ggtggcctaa ctacggctac 3120 actagaagga cagtatttgg tatctgcgctctgctgaagc cagttacctt cggaaaaaga 3180 gttggtagct cttgatccgg caaacaaaccaccgctggta gcggtggttt ttttgtttgc 3240 aagcagcaga ttacgcgcag aaaaaaaggatctcaagaag atcctttgat cttttctacg 3300 gggtctgacg ctcagtggaa cgaaaactcacgttaaggga ttttggtcat gagattatca 3360 aaaaggatct tcacctagat ccttttaaattaaaaatgaa gttttaaatc aatctaaagt 3420 atatatgagt aaacttggtc tgacagttaccaatgcttaa tcagtgaggc acctatctca 3480 gcgatctgtc tatttcgttc atccatagttgcctgactcc ccgtcgtgta gataactacg 3540 atacgggagg gcttaccatc tggccccagtgctgcaatga taccgcgaga cccacgctca 3600 ccggctccag atttatcagc aataaaccagccagccggaa gggccgagcg cagaagtggt 3660 cctgcaactt tatccgcctc catccagtctattaattgtt gccgggaagc tagagtaagt 3720 agttcgccag ttaatagttt gcgcaacgttgttgccattg ctacaggcat cgtggtgtca 3780 cgctcgtcgt ttggtatggc ttcattcagctccggttccc aacgatcaag gcgagttaca 3840 tgatccccca tgttgtgcaa aaaagcggttagctccttcg gtcctccgat cgttgtcaga 3900 agtaagttgg ccgcagtgtt atcactcatggttatggcag cactgcataa ttctcttact 3960 gtcatgccat ccgtaagatg cttttctgtgactggtgagt actcaaccaa gtcattctga 4020 gaatagtgta tgcggcgacc gagttgctcttgcccggcgt caatacggga taataccgcg 4080 ccacatagca gaactttaaa agtgctcatcattggaaaac gttcttcggg gcgaaaactc 4140 tcaaggatct taccgctgtt gagatccagttcgatgtaac ccactcgtgc acccaactga 4200 tcttcagcat cttttacttt caccagcgtttctgggtgag caaaaacagg aaggcaaaat 4260 gccgcaaaaa agggaataag ggcgacacggaaatgttgaa tactcatact cttccttttt 4320 caatattatt gaagcattta tcagggttattgtctcatga gcggatacat atttgaatgt 4380 atttagaaaa ataaacaaat aggggttccgcgcacatttc cccgaaaagt gccacctgac 4440 gtctaagaaa ccattattat catgacattaacctataaaa ataggcgtat cacgaggccc 4500 tttcgtc 4507 17 5410 DNA UnknownSequence represents a plant promoter-terminator expression cassette invector pUC19 17 ttttggaaat gatttgcatg gaagccatgt gtaaaaccat gacatccacttggaggatgc 60 aataatgaag aaaactacaa atttacatgc aactagttat gcatgtagtctatataatga 120 ggattttgca atactttcat tcatacacac tcactaagtt ttacacgattataatttctt 180 catagccagc ggatccgata tcgggcccgc tagcgttaac cctgctttaatgagatatgc 240 gagacgccta tgatcgcatg atatttgctt tcaattctgt tgtgcacgttgtaaaaaacc 300 tgagcatgtg tagctcagat ccttaccgcc ggtttcggtt cattctaatgaatatatcac 360 ccgttactat cgtattttta tgaataatat tctccgttca atttactgattgtccgtcga 420 gcaaatttac acattgccac taaacgtcta aacccttgta atttgtttttgttttactat 480 gtgtgttatg tatttgattt gcgataaatt tttatatttg gtactaaatttataacacct 540 tttatgctaa cgtttgccaa cacttagcaa tttgcaagtt gattaattgattctaaatta 600 tttttgtctt ctaaatacat atactaatca actggaaatg taaatatttgctaatatttc 660 tactatagga gaattaaagt gagtgaatat ggtaccacaa ggtttggagatttaattgtt 720 gcaatgctgc atggatggca tatacaccaa acattcaata attcttgaggataataatgg 780 taccacacaa gatttgaggt gcatgaacgt cacgtggaca aaaggtttagtaatttttca 840 agacaacaat gttaccacac acaagttttg aggtgcatgc atggatgccctgtggaaagt 900 ttaaaaatat tttggaaatg atttgcatgg aagccatgtg taaaaccatgacatccactt 960 ggaggatgca ataatgaaga aaactacaaa tttacatgca actagttatgcatgtagtct 1020 atataatgag gattttgcaa tactttcatt catacacact cactaagttttacacgatta 1080 taatttcttc atagccagca gatctgccgg catcgatccc gggccatggcctgctttaat 1140 gagatatgcg agacgcctat gatcgcatga tatttgcttt caattctgttgtgcacgttg 1200 taaaaaacct gagcatgtgt agctcagatc cttaccgccg gtttcggttcattctaatga 1260 atatatcacc cgttactatc gtatttttat gaataatatt ctccgttcaatttactgatt 1320 gtccgtcgac gagctcggcg cgccaagctt ggcgtaatca tggtcatagctgtttcctgt 1380 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagcataaagtgtaa 1440 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgctcactgcccgc 1500 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaacgcgcggggag 1560 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgctgcgctcggt 1620 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggttatccacaga 1680 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaaggccaggaaccg 1740 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacgagcatcacaa 1800 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagataccaggcgtt 1860 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgcttaccggatacct 1920 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgctgtaggtatct 1980 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaaccccccgttcagcc 2040 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaagacacgactt 2100 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatgtaggcggtgc 2160 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacagtatttggtat 2220 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctcttgatccggcaa 2280 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagattacgcgcagaaa 2340 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctcagtggaacga 2400 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttcacctagatcct 2460 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaacttggtctga 2520 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctatttcgttcatc 2580 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggcttaccatctgg 2640 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatttatcagcaat 2700 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttatccgcctccat 2760 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagttaatagtttgcg 2820 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttggtatggcttc 2880 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgttgtgcaaaaa 2940 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccgcagtgttatc 3000 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccgtaagatgctt 3060 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgcggcgaccgag 3120 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaactttaaaagt 3180 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttaccgctgttgag 3240 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatcttttactttcac 3300 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagggaataagggc 3360 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaagcatttatca 3420 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaataaacaaatagg 3480 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaaccattattatcat 3540 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgcgtttcggtga 3600 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagcttgtctgtaagc 3660 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcgggtgtcgggg 3720 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatatgcggtgtga 3780 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgccattcaggct 3840 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgccagctggcgaa 3900 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttcccagtcacgacg 3960 ttgtaaaacg acggccagtg aattcggcgc gccgagctcc tcgagcaaatttacacattg 4020 ccactaaacg tctaaaccct tgtaatttgt ttttgtttta ctatgtgtgttatgtatttg 4080 atttgcgata aatttttata tttggtacta aatttataac accttttatgctaacgtttg 4140 ccaacactta gcaatttgca agttgattaa ttgattctaa attatttttgtcttctaaat 4200 acatatacta atcaactgga aatgtaaata tttgctaata tttctactataggagaatta 4260 aagtgagtga atatggtacc acaaggtttg gagatttaat tgttgcaatgctgcatggat 4320 ggcatataca ccaaacattc aataattctt gaggataata atggtaccacacaagatttg 4380 aggtgcatga acgtcacgtg gacaaaaggt ttagtaattt ttcaagacaacaatgttacc 4440 acacacaagt tttgaggtgc atgcatggat gccctgtgga aagtttaaaaatattttgga 4500 aatgatttgc atggaagcca tgtgtaaaac catgacatcc acttggaggatgcaataatg 4560 aagaaaacta caaatttaca tgcaactagt tatgcatgta gtctatataatgaggatttt 4620 gcaatacttt cattcataca cactcactaa gttttacacg attataatttcttcatagcc 4680 agcccaccgc ggtgggcggc cgcctgcagt ctagaaggcc tcctgctttaatgagatatg 4740 cgagacgcct atgatcgcat gatatttgct ttcaattctg ttgtgcacgttgtaaaaaac 4800 ctgagcatgt gtagctcaga tccttaccgc cggtttcggt tcattctaatgaatatatca 4860 cccgttacta tcgtattttt atgaataata ttctccgttc aatttactgattgtccgtcg 4920 agcaaattta cacattgcca ctaaacgtct aaacccttgt aatttgtttttgttttacta 4980 tgtgtgttat gtatttgatt tgcgataaat ttttatattt ggtactaaatttataacacc 5040 ttttatgcta acgtttgcca acacttagca atttgcaagt tgattaattgattctaaatt 5100 atttttgtct tctaaataca tatactaatc aactggaaat gtaaatatttgctaatattt 5160 ctactatagg agaattaaag tgagtgaata tggtaccaca aggtttggagatttaattgt 5220 tgcaatgctg catggatggc atatacacca aacattcaat aattcttgaggataataatg 5280 gtaccacaca agatttgagg tgcatgaacg tcacgtggac aaaaggtttagtaatttttc 5340 aagacaacaa tgttaccaca cacaagtttt gaggtgcatg catggatgccctgtggaaag 5400 tttaaaaata 5410 18 648 DNA Phaeodactylum tricornutum CDS(1)..(648) . 18 tgg tgg aaa aac aag cac aac gga cac cac gcc gtc ccc aacctc cac 48 Trp Trp Lys Asn Lys His Asn Gly His His Ala Val Pro Asn LeuHis 1 5 10 15 tgc tcc tcc gca gtc gcg caa gat ggg gac ccg gac atc gatacc atg 96 Cys Ser Ser Ala Val Ala Gln Asp Gly Asp Pro Asp Ile Asp ThrMet 20 25 30 ccc ctt ctc gcc tgg tcc gtc cag caa gcc cag tct tac cgg gaactc 144 Pro Leu Leu Ala Trp Ser Val Gln Gln Ala Gln Ser Tyr Arg Glu Leu35 40 45 caa gcc gac gga aag gat tcg ggt ttg gtc aag ttc atg atc cgt aac192 Gln Ala Asp Gly Lys Asp Ser Gly Leu Val Lys Phe Met Ile Arg Asn 5055 60 caa tcc tac ttt tac ttt ccc atc ttg ttg ctc gcc cgc ctg tcg tgg240 Gln Ser Tyr Phe Tyr Phe Pro Ile Leu Leu Leu Ala Arg Leu Ser Trp 6570 75 80 ttg aac gag tcc ttc aag tgc gcc ttt ggg ctt gga gct gcg tcg gag288 Leu Asn Glu Ser Phe Lys Cys Ala Phe Gly Leu Gly Ala Ala Ser Glu 8590 95 aac gct gct ctc gaa ctc aag gcc aag ggt ctt cag tac ccc ctt ttg336 Asn Ala Ala Leu Glu Leu Lys Ala Lys Gly Leu Gln Tyr Pro Leu Leu 100105 110 gaa aag gct ggc atc ctg ctg cac tac gct tgg atg ctt aca gtt tcg384 Glu Lys Ala Gly Ile Leu Leu His Tyr Ala Trp Met Leu Thr Val Ser 115120 125 tcc ggc ttt gga cgc ttc tcg ttc gcg tac acc gca ttt tac ttt cta432 Ser Gly Phe Gly Arg Phe Ser Phe Ala Tyr Thr Ala Phe Tyr Phe Leu 130135 140 acc gcg acc gcg tcc tgt gga ttc ttg ctc gcc att gtc ttt ggc ctc480 Thr Ala Thr Ala Ser Cys Gly Phe Leu Leu Ala Ile Val Phe Gly Leu 145150 155 160 ggc cac aac ggc atg gcc acc tac aat gcc gac gcc cgt ccg gacttc 528 Gly His Asn Gly Met Ala Thr Tyr Asn Ala Asp Ala Arg Pro Asp Phe165 170 175 tgg aag ctc caa gtc acc acg act cgc aac gtc acg ggc gga cacggt 576 Trp Lys Leu Gln Val Thr Thr Thr Arg Asn Val Thr Gly Gly His Gly180 185 190 ttc ccc caa gcc ttt gtc gac tgg ttc tgt ggt ggc ctc cag taccaa 624 Phe Pro Gln Ala Phe Val Asp Trp Phe Cys Gly Gly Leu Gln Tyr Gln195 200 205 gtc gac cac cac tta ttc ccc agc 648 Val Asp His His Leu PhePro Ser 210 215 19 216 PRT Phaeodactylum tricornutum 19 Trp Trp Lys AsnLys His Asn Gly His His Ala Val Pro Asn Leu His 1 5 10 15 Cys Ser SerAla Val Ala Gln Asp Gly Asp Pro Asp Ile Asp Thr Met 20 25 30 Pro Leu LeuAla Trp Ser Val Gln Gln Ala Gln Ser Tyr Arg Glu Leu 35 40 45 Gln Ala AspGly Lys Asp Ser Gly Leu Val Lys Phe Met Ile Arg Asn 50 55 60 Gln Ser TyrPhe Tyr Phe Pro Ile Leu Leu Leu Ala Arg Leu Ser Trp 65 70 75 80 Leu AsnGlu Ser Phe Lys Cys Ala Phe Gly Leu Gly Ala Ala Ser Glu 85 90 95 Asn AlaAla Leu Glu Leu Lys Ala Lys Gly Leu Gln Tyr Pro Leu Leu 100 105 110 GluLys Ala Gly Ile Leu Leu His Tyr Ala Trp Met Leu Thr Val Ser 115 120 125Ser Gly Phe Gly Arg Phe Ser Phe Ala Tyr Thr Ala Phe Tyr Phe Leu 130 135140 Thr Ala Thr Ala Ser Cys Gly Phe Leu Leu Ala Ile Val Phe Gly Leu 145150 155 160 Gly His Asn Gly Met Ala Thr Tyr Asn Ala Asp Ala Arg Pro AspPhe 165 170 175 Trp Lys Leu Gln Val Thr Thr Thr Arg Asn Val Thr Gly GlyHis Gly 180 185 190 Phe Pro Gln Ala Phe Val Asp Trp Phe Cys Gly Gly LeuGln Tyr Gln 195 200 205 Val Asp His His Leu Phe Pro Ser 210 215 20 12093DNA Unknown Plant expression vector with a promoter- terminatorexpression cassette 20 gatctggcgc cggccagcga gacgagcaag attggccgccgcccgaaacg atccgacagc 60 gcgcccagca caggtgcgca ggcaaattgc accaacgcatacagcgccag cagaatgcca 120 tagtgggcgg tgacgtcgtt cgagtgaacc agatcgcgcaggaggcccgg cagcaccggc 180 ataatcaggc cgatgccgac agcgtcgagc gcgacagtgctcagaattac gatcaggggt 240 atgttgggtt tcacgtctgg cctccggacc agcctccgctggtccgattg aacgcgcgga 300 ttctttatca ctgataagtt ggtggacata ttatgtttatcagtgataaa gtgtcaagca 360 tgacaaagtt gcagccgaat acagtgatcc gtgccgccctggacctgttg aacgaggtcg 420 gcgtagacgg tctgacgaca cgcaaactgg cggaacggttgggggttcag cagccggcgc 480 tttactggca cttcaggaac aagcgggcgc tgctcgacgcactggccgaa gccatgctgg 540 cggagaatca tacgcattcg gtgccgagag ccgacgacgactggcgctca tttctgatcg 600 ggaatgcccg cagcttcagg caggcgctgc tcgcctaccgcgatggcgcg cgcatccatg 660 ccggcacgcg accgggcgca ccgcagatgg aaacggccgacgcgcagctt cgcttcctct 720 gcgaggcggg tttttcggcc ggggacgccg tcaatgcgctgatgacaatc agctacttca 780 ctgttggggc cgtgcttgag gagcaggccg gcgacagcgatgccggcgag cgcggcggca 840 ccgttgaaca ggctccgctc tcgccgctgt tgcgggccgcgatagacgcc ttcgacgaag 900 ccggtccgga cgcagcgttc gagcagggac tcgcggtgattgtcgatgga ttggcgaaaa 960 ggaggctcgt tgtcaggaac gttgaaggac cgagaaagggtgacgattga tcaggaccgc 1020 tgccggagcg caacccactc actacagcag agccatgtagacaacatccc ctcccccttt 1080 ccaccgcgtc agacgcccgt agcagcccgc tacgggctttttcatgccct gccctagcgt 1140 ccaagcctca cggccgcgct cggcctctct ggcggccttctggcgctctt ccgcttcctc 1200 gctcactgac tcgctgcgct cggtcgttcg gctgcggcgagcggtatcag ctcactcaaa 1260 ggcggtaata cggttatcca cagaatcagg ggataacgcaggaaagaaca tgtgagcaaa 1320 aggccagcaa aaggccagga accgtaaaaa ggccgcgttgctggcgtttt tccataggct 1380 ccgcccccct gacgagcatc acaaaaatcg acgctcaagtcagaggtggc gaaacccgac 1440 aggactataa agataccagg cgtttccccc tggaagctccctcgtgcgct ctcctgttcc 1500 gaccctgccg cttaccggat acctgtccgc ctttctcccttcgggaagcg tggcgctttt 1560 ccgctgcata accctgcttc ggggtcatta tagcgattttttcggtatat ccatcctttt 1620 tcgcacgata tacaggattt tgccaaaggg ttcgtgtagactttccttgg tgtatccaac 1680 ggcgtcagcc gggcaggata ggtgaagtag gcccacccgcgagcgggtgt tccttcttca 1740 ctgtccctta ttcgcacctg gcggtgctca acgggaatcctgctctgcga ggctggccgg 1800 ctaccgccgg cgtaacagat gagggcaagc ggatggctgatgaaaccaag ccaaccagga 1860 agggcagccc acctatcaag gtgtactgcc ttccagacgaacgaagagcg attgaggaaa 1920 aggcggcggc ggccggcatg agcctgtcgg cctacctgctggccgtcggc cagggctaca 1980 aaatcacggg cgtcgtggac tatgagcacg tccgcgagctggcccgcatc aatggcgacc 2040 tgggccgcct gggcggcctg ctgaaactct ggctcaccgacgacccgcgc acggcgcggt 2100 tcggtgatgc cacgatcctc gccctgctgg cgaagatcgaagagaagcag gacgagcttg 2160 gcaaggtcat gatgggcgtg gtccgcccga gggcagagccatgacttttt tagccgctaa 2220 aacggccggg gggtgcgcgt gattgccaag cacgtccccatgcgctccat caagaagagc 2280 gacttcgcgg agctggtgaa gtacatcacc gacgagcaaggcaagaccga gcgcctttgc 2340 gacgctcacc gggctggttg ccctcgccgc tgggctggcggccgtctatg gccctgcaaa 2400 cgcgccagaa acgccgtcga agccgtgtgc gagacaccgcggccgccggc gttgtggata 2460 cctcgcggaa aacttggccc tcactgacag atgaggggcggacgttgaca cttgaggggc 2520 cgactcaccc ggcgcggcgt tgacagatga ggggcaggctcgatttcggc cggcgacgtg 2580 gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattttacgcgagtt tcccacagat 2640 gatgtggaca agcctgggga taagtgccct gcggtattgacacttgaggg gcgcgactac 2700 tgacagatga ggggcgcgat ccttgacact tgaggggcagagtgctgaca gatgaggggc 2760 gcacctattg acatttgagg ggctgtccac aggcagaaaatccagcattt gcaagggttt 2820 ccgcccgttt ttcggccacc gctaacctgt cttttaacctgcttttaaac caatatttat 2880 aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtgaccgcgcacgc cgaagggggg 2940 tgccccccct tctcgaaccc tcccggcccg ctaacgcgggcctcccatcc ccccaggggc 3000 tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggcagcgctggca gtccttgcca 3060 ttgccgggat cggggcagta acgggatggg cgatcagcccgagcgcgacg cccggaagca 3120 ttgacgtgcc gcaggtgctg gcatcgacat tcagcgaccaggtgccgggc agtgagggcg 3180 gcggcctggg tggcggcctg cccttcactt cggccgtcggggcattcacg gacttcatgg 3240 cggggccggc aatttttacc ttgggcattc ttggcatagtggtcgcgggt gccgtgctcg 3300 tgttcggggg tgcgataaac ccagcgaacc atttgaggtgataggtaaga ttataccgag 3360 gtatgaaaac gagaattgga cctttacaga attactctatgaagcgccat atttaaaaag 3420 ctaccaagac gaagaggatg aagaggatga ggaggcagattgccttgaat atattgacaa 3480 tactgataag ataatatatc ttttatatag aagatatcgccgtatgtaag gatttcaggg 3540 ggcaaggcat aggcagcgcg cttatcaata tatctatagaatgggcaaag cataaaaact 3600 tgcatggact aatgcttgaa acccaggaca ataaccttatagcttgtaaa ttctatcata 3660 attgggtaat gactccaact tattgatagt gttttatgttcagataatgc ccgatgactt 3720 tgtcatgcag ctccaccgat tttgagaacg acagcgacttccgtcccagc cgtgccaggt 3780 gctgcctcag attcaggtta tgccgctcaa ttcgctgcgtatatcgcttg ctgattacgt 3840 gcagctttcc cttcaggcgg gattcataca gcggccagccatccgtcatc catatcacca 3900 cgtcaaaggg tgacagcagg ctcataagac gccccagcgtcgccatagtg cgttcaccga 3960 atacgtgcgc aacaaccgtc ttccggagac tgtcatacgcgtaaaacagc cagcgctggc 4020 gcgatttagc cccgacatag ccccactgtt cgtccatttccgcgcagacg atgacgtcac 4080 tgcccggctg tatgcgcgag gttaccgact gcggcctgagttttttaagt gacgtaaaat 4140 cgtgttgagg ccaacgccca taatgcgggc tgttgcccggcatccaacgc cattcatggc 4200 catatcaatg attttctggt gcgtaccggg ttgagaagcggtgtaagtga actgcagttg 4260 ccatgtttta cggcagtgag agcagagata gcgctgatgtccggcggtgc ttttgccgtt 4320 acgcaccacc ccgtcagtag ctgaacagga gggacagctgatagacacag aagccactgg 4380 agcacctcaa aaacaccatc atacactaaa tcagtaagttggcagcatca cccataattg 4440 tggtttcaaa atcggctccg tcgatactat gttatacgccaactttgaaa acaactttga 4500 aaaagctgtt ttctggtatt taaggtttta gaatgcaaggaacagtgaat tggagttcgt 4560 cttgttataa ttagcttctt ggggtatctt taaatactgtagaaaagagg aaggaaataa 4620 taaatggcta aaatgagaat atcaccggaa ttgaaaaaactgatcgaaaa ataccgctgc 4680 gtaaaagata cggaaggaat gtctcctgct aaggtatataagctggtggg agaaaatgaa 4740 aacctatatt taaaaatgac ggacagccgg tataaagggaccacctatga tgtggaacgg 4800 gaaaaggaca tgatgctatg gctggaagga aagctgcctgttccaaaggt cctgcacttt 4860 gaacggcatg atggctggag caatctgctc atgagtgaggccgatggcgt cctttgctcg 4920 gaagagtatg aagatgaaca aagccctgaa aagattatcgagctgtatgc ggagtgcatc 4980 aggctctttc actccatcga catatcggat tgtccctatacgaatagctt agacagccgc 5040 ttagccgaat tggattactt actgaataac gatctggccgatgtggattg cgaaaactgg 5100 gaagaagaca ctccatttaa agatccgcgc gagctgtatgattttttaaa gacggaaaag 5160 cccgaagagg aacttgtctt ttcccacggc gacctgggagacagcaacat ctttgtgaaa 5220 gatggcaaag taagtggctt tattgatctt gggagaagcggcagggcgga caagtggtat 5280 gacattgcct tctgcgtccg gtcgatcagg gaggatatcggggaagaaca gtatgtcgag 5340 ctattttttg acttactggg gatcaagcct gattgggagaaaataaaata ttatatttta 5400 ctggatgaat tgttttagta cctagatgtg gcgcaacgatgccggcgaca agcaggagcg 5460 caccgacttc ttccgcatca agtgttttgg ctctcaggccgaggcccacg gcaagtattt 5520 gggcaagggg tcgctggtat tcgtgcaggg caagattcggaataccaagt acgagaagga 5580 cggccagacg gtctacggga ccgacttcat tgccgataaggtggattatc tggacaccaa 5640 ggcaccaggc gggtcaaatc aggaataagg gcacattgccccggcgtgag tcggggcaat 5700 cccgcaagga gggtgaatga atcggacgtt tgaccggaaggcatacaggc aagaactgat 5760 cgacgcgggg ttttccgccg aggatgccga aaccatcgcaagccgcaccg tcatgcgtgc 5820 gccccgcgaa accttccagt ccgtcggctc gatggtccagcaagctacgg ccaagatcga 5880 gcgcgacagc gtgcaactgg ctccccctgc cctgcccgcgccatcggccg ccgtggagcg 5940 ttcgcgtcgt ctcgaacagg aggcggcagg tttggcgaagtcgatgacca tcgacacgcg 6000 aggaactatg acgaccaaga agcgaaaaac cgccggcgaggacctggcaa aacaggtcag 6060 cgaggccaag caggccgcgt tgctgaaaca cacgaagcagcagatcaagg aaatgcagct 6120 ttccttgttc gatattgcgc cgtggccgga cacgatgcgagcgatgccaa acgacacggc 6180 ccgctctgcc ctgttcacca cgcgcaacaa gaaaatcccgcgcgaggcgc tgcaaaacaa 6240 ggtcattttc cacgtcaaca aggacgtgaa gatcacctacaccggcgtcg agctgcgggc 6300 cgacgatgac gaactggtgt ggcagcaggt gttggagtacgcgaagcgca cccctatcgg 6360 cgagccgatc accttcacgt tctacgagct ttgccaggacctgggctggt cgatcaatgg 6420 ccggtattac acgaaggccg aggaatgcct gtcgcgcctacaggcgacgg cgatgggctt 6480 cacgtccgac cgcgttgggc acctggaatc ggtgtcgctgctgcaccgct tccgcgtcct 6540 ggaccgtggc aagaaaacgt cccgttgcca ggtcctgatcgacgaggaaa tcgtcgtgct 6600 gtttgctggc gaccactaca cgaaattcat atgggagaagtaccgcaagc tgtcgccgac 6660 ggcccgacgg atgttcgact atttcagctc gcaccgggagccgtacccgc tcaagctgga 6720 aaccttccgc ctcatgtgcg gatcggattc cacccgcgtgaagaagtggc gcgagcaggt 6780 cggcgaagcc tgcgaagagt tgcgaggcag cggcctggtggaacacgcct gggtcaatga 6840 tgacctggtg cattgcaaac gctagggcct tgtggggtcagttccggctg ggggttcagc 6900 agccagcgct ttactggcat ttcaggaaca agcgggcactgctcgacgca cttgcttcgc 6960 tcagtatcgc tcgggacgca cggcgcgctc tacgaactgccgataaacag aggattaaaa 7020 ttgacaattg tgattaaggc tcagattcga cggcttggagcggccgacgt gcaggatttc 7080 cgcgagatcc gattgtcggc cctgaagaaa gctccagagatgttcgggtc cgtttacgag 7140 cacgaggaga aaaagcccat ggaggcgttc gctgaacggttgcgagatgc cgtggcattc 7200 ggcgcctaca tcgacggcga gatcattggg ctgtcggtcttcaaacagga ggacggcccc 7260 aaggacgctc acaaggcgca tctgtccggc gttttcgtggagcccgaaca gcgaggccga 7320 ggggtcgccg gtatgctgct gcgggcgttg ccggcgggtttattgctcgt gatgatcgtc 7380 cgacagattc caacgggaat ctggtggatg cgcatcttcatcctcggcgc acttaatatt 7440 tcgctattct ggagcttgtt gtttatttcg gtctaccgcctgccgggcgg ggtcgcggcg 7500 acggtaggcg ctgtgcagcc gctgatggtc gtgttcatctctgccgctct gctaggtagc 7560 ccgatacgat tgatggcggt cctgggggct atttgcggaactgcgggcgt ggcgctgttg 7620 gtgttgacac caaacgcagc gctagatcct gtcggcgtcgcagcgggcct ggcgggggcg 7680 gtttccatgg cgttcggaac cgtgctgacc cgcaagtggcaacctcccgt gcctctgctc 7740 acctttaccg cctggcaact ggcggccgga ggacttctgctcgttccagt agctttagtg 7800 tttgatccgc caatcccgat gcctacagga accaatgttctcggcctggc gtggctcggc 7860 ctgatcggag cgggtttaac ctacttcctt tggttccgggggatctcgcg actcgaacct 7920 acagttgttt ccttactggg ctttctcagc cccagatctggggtcgatca gccggggatg 7980 catcaggccg acagtcggaa cttcgggtcc ccgacctgtaccattcggtg agcaatggat 8040 aggggagttg atatcgtcaa cgttcacttc taaagaaatagcgccactca gcttcctcag 8100 cggctttatc cagcgatttc ctattatgtc ggcatagttctcaagatcga cagcctgtca 8160 cggttaagcg agaaatgaat aagaaggctg ataattcggatctctgcgag ggagatgata 8220 tttgatcaca ggcagcaacg ctctgtcatc gttacaatcaacatgctacc ctccgcgaga 8280 tcatccgtgt ttcaaacccg gcagcttagt tgccgttcttccgaatagca tcggtaacat 8340 gagcaaagtc tgccgcctta caacggctct cccgctgacgccgtcccgga ctgatgggct 8400 gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcggggagctgtt ggctggctgg 8460 tggcaggata tattgtggtg taaacaaatt gacgcttagacaacttaata acacattgcg 8520 gacgttttta atgtactggg gtggtttttc ttttcaccagtgagacgggc aacagctgat 8580 tgcccttcac cgcctggccc tgagagagtt gcagcaagcggtccacgctg gtttgcccca 8640 gcaggcgaaa atcctgtttg atggtggttc cgaaatcggcaaaatccctt ataaatcaaa 8700 agaatagccc gagatagggt tgagtgttgt tccagtttggaacaagagtc cactattaaa 8760 gaacgtggac tccaacgtca aagggcgaaa aaccgtctatcagggcgatg gcccactacg 8820 tgaaccatca cccaaatcaa gttttttggg gtcgaggtgccgtaaagcac taaatcggaa 8880 ccctaaaggg agcccccgat ttagagcttg acggggaaagccggcgaacg tggcgagaaa 8940 ggaagggaag aaagcgaaag gagcgggcgc cattcaggctgcgcaactgt tgggaagggc 9000 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaaagggggatgt gctgcaaggc 9060 gattaagttg ggtaacgcca gggttttccc agtcacgacgttgtaaaacg acggccagtg 9120 aattaattcc catcttgaaa gaaatatagt ttaaatatttattgataaaa taacaagtca 9180 ggtattatag tccaagcaaa aacataaatt tattgatgcaagtttaaatt cagaaatatt 9240 tcaataactg attatatcag ctggtacatt gccgtagatgaaagactgag tgcgatatta 9300 tgtgtaatac ataaattgat gatatagcta gcttagctcatcgggggatc cgtcgaagct 9360 agcttgggtc ccgctcagaa gaactcgtca agaaggcgatagaaggcgat gcgctgcgaa 9420 tcgggagcgg cgataccgta aagcacgagg aagcggtcagcccattcgcc gccaagctct 9480 tcagcaatat cacgggtagc caacgctatg tcctgatagcggtccgccac acccagccgg 9540 ccacagtcga tgaatccaga aaagcggcca ttttccaccatgatattcgg caagcaggca 9600 tcgccatggg tcacgacgag atcctcgccg tcgggcatgcgcgccttgag cctggcgaac 9660 agttcggctg gcgcgagccc ctgatgctct tcgtccagatcatcctgatc gacaagaccg 9720 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcgcttggtggtc gaatgggcag 9780 gtagccggat caagcgtatg cagccgccgc attgcatcagccatgatgga tactttctcg 9840 gcaggagcaa ggtgagatga caggagatcc tgccccggcacttcgcccaa tagcagccag 9900 tcccttcccg cttcagtgac aacgtcgagc acagctgcgcaaggaacgcc cgtcgtggcc 9960 agccacgata gccgcgctgc ctcgtcctgc agttcattcagggcaccgga caggtcggtc 10020 ttgacaaaaa gaaccgggcg cccctgcgct gacagccggaacacggcggc atcagagcag 10080 ccgattgtct gttgtgccca gtcatagccg aatagcctctccacccaagc ggccggagaa 10140 cctgcgtgca atccatcttg ttcaatccaa gctcccatgggccctcgact agagtcgaga 10200 tctggattga gagtgaatat gagactctaa ttggataccgaggggaattt atggaacgtc 10260 agtggagcat ttttgacaag aaatatttgc tagctgatagtgaccttagg cgacttttga 10320 acgcgcaata atggtttctg acgtatgtgc ttagctcattaaactccaga aacccgcggc 10380 tgagtggctc cttcaacgtt gcggttctgt cagttccaaacgtaaaacgg cttgtcccgc 10440 gtcatcggcg ggggtcataa cgtgactccc ttaattctccgctcatgatc ttgatcccct 10500 gcgccatcag atccttggcg gcaagaaagc catccagtttactttgcagg gcttcccaac 10560 cttaccagag ggcgccccag ctggcaattc cggttcgcttgctgtccata aaaccgccca 10620 gtctagctat cgccatgtaa gcccactgca agctacctgctttctctttg cgcttgcgtt 10680 ttcccttgtc cagatagccc agtagctgac attcatccggggtcagcacc gtttctgcgg 10740 actggctttc tacgtgttcc gcttccttta gcagcccttgcgccctgagt gcttgcggca 10800 gcgtgaagct tgcatgcctg caggtcgacg gcgcgccgagctcctcgagc aaatttacac 10860 attgccacta aacgtctaaa cccttgtaat ttgtttttgttttactatgt gtgttatgta 10920 tttgatttgc gataaatttt tatatttggt actaaatttataacaccttt tatgctaacg 10980 tttgccaaca cttagcaatt tgcaagttga ttaattgattctaaattatt tttgtcttct 11040 aaatacatat actaatcaac tggaaatgta aatatttgctaatatttcta ctataggaga 11100 attaaagtga gtgaatatgg taccacaagg tttggagatttaattgttgc aatgctgcat 11160 ggatggcata tacaccaaac attcaataat tcttgaggataataatggta ccacacaaga 11220 tttgaggtgc atgaacgtca cgtggacaaa aggtttagtaatttttcaag acaacaatgt 11280 taccacacac aagttttgag gtgcatgcat ggatgccctgtggaaagttt aaaaatattt 11340 tggaaatgat ttgcatggaa gccatgtgta aaaccatgacatccacttgg aggatgcaat 11400 aatgaagaaa actacaaatt tacatgcaac tagttatgcatgtagtctat ataatgagga 11460 ttttgcaata ctttcattca tacacactca ctaagttttacacgattata atttcttcat 11520 agccagccca ccgcggtggg cggccgcctg cagtctagaaggcctcctgc tttaatgaga 11580 tatgcgagac gcctatgatc gcatgatatt tgctttcaattctgttgtgc acgttgtaaa 11640 aaacctgagc atgtgtagct cagatcctta ccgccggtttcggttcattc taatgaatat 11700 atcacccgtt actatcgtat ttttatgaat aatattctccgttcaattta ctgattgtcc 11760 gtcgacgaat tcgagctcgg cgcgcctcta gaggatcgatgaattcagat cggctgagtg 11820 gctccttcaa cgttgcggtt ctgtcagttc caaacgtaaaacggcttgtc ccgcgtcatc 11880 ggcgggggtc ataacgtgac tcccttaatt ctccgctcatgatcagattg tcgtttcccg 11940 ccttcagttt aaactatcag tgtttgacag gatatattggcgggtaaacc taagagaaaa 12000 gagcgtttat tagaataatc ggatatttaa aagggcgtgaaaaggtttat ccttcgtcca 12060 tttgtatgtg catgccaacc acagggttcc cca 1209321 12085 DNA Unknown Plant expression vector with a promoter- terminatorexpression cassette 21 gatctggcgc cggccagcga gacgagcaag attggccgccgcccgaaacg atccgacagc 60 gcgcccagca caggtgcgca ggcaaattgc accaacgcatacagcgccag cagaatgcca 120 tagtgggcgg tgacgtcgtt cgagtgaacc agatcgcgcaggaggcccgg cagcaccggc 180 ataatcaggc cgatgccgac agcgtcgagc gcgacagtgctcagaattac gatcaggggt 240 atgttgggtt tcacgtctgg cctccggacc agcctccgctggtccgattg aacgcgcgga 300 ttctttatca ctgataagtt ggtggacata ttatgtttatcagtgataaa gtgtcaagca 360 tgacaaagtt gcagccgaat acagtgatcc gtgccgccctggacctgttg aacgaggtcg 420 gcgtagacgg tctgacgaca cgcaaactgg cggaacggttgggggttcag cagccggcgc 480 tttactggca cttcaggaac aagcgggcgc tgctcgacgcactggccgaa gccatgctgg 540 cggagaatca tacgcattcg gtgccgagag ccgacgacgactggcgctca tttctgatcg 600 ggaatgcccg cagcttcagg caggcgctgc tcgcctaccgcgatggcgcg cgcatccatg 660 ccggcacgcg accgggcgca ccgcagatgg aaacggccgacgcgcagctt cgcttcctct 720 gcgaggcggg tttttcggcc ggggacgccg tcaatgcgctgatgacaatc agctacttca 780 ctgttggggc cgtgcttgag gagcaggccg gcgacagcgatgccggcgag cgcggcggca 840 ccgttgaaca ggctccgctc tcgccgctgt tgcgggccgcgatagacgcc ttcgacgaag 900 ccggtccgga cgcagcgttc gagcagggac tcgcggtgattgtcgatgga ttggcgaaaa 960 ggaggctcgt tgtcaggaac gttgaaggac cgagaaagggtgacgattga tcaggaccgc 1020 tgccggagcg caacccactc actacagcag agccatgtagacaacatccc ctcccccttt 1080 ccaccgcgtc agacgcccgt agcagcccgc tacgggctttttcatgccct gccctagcgt 1140 ccaagcctca cggccgcgct cggcctctct ggcggccttctggcgctctt ccgcttcctc 1200 gctcactgac tcgctgcgct cggtcgttcg gctgcggcgagcggtatcag ctcactcaaa 1260 ggcggtaata cggttatcca cagaatcagg ggataacgcaggaaagaaca tgtgagcaaa 1320 aggccagcaa aaggccagga accgtaaaaa ggccgcgttgctggcgtttt tccataggct 1380 ccgcccccct gacgagcatc acaaaaatcg acgctcaagtcagaggtggc gaaacccgac 1440 aggactataa agataccagg cgtttccccc tggaagctccctcgtgcgct ctcctgttcc 1500 gaccctgccg cttaccggat acctgtccgc ctttctcccttcgggaagcg tggcgctttt 1560 ccgctgcata accctgcttc ggggtcatta tagcgattttttcggtatat ccatcctttt 1620 tcgcacgata tacaggattt tgccaaaggg ttcgtgtagactttccttgg tgtatccaac 1680 ggcgtcagcc gggcaggata ggtgaagtag gcccacccgcgagcgggtgt tccttcttca 1740 ctgtccctta ttcgcacctg gcggtgctca acgggaatcctgctctgcga ggctggccgg 1800 ctaccgccgg cgtaacagat gagggcaagc ggatggctgatgaaaccaag ccaaccagga 1860 agggcagccc acctatcaag gtgtactgcc ttccagacgaacgaagagcg attgaggaaa 1920 aggcggcggc ggccggcatg agcctgtcgg cctacctgctggccgtcggc cagggctaca 1980 aaatcacggg cgtcgtggac tatgagcacg tccgcgagctggcccgcatc aatggcgacc 2040 tgggccgcct gggcggcctg ctgaaactct ggctcaccgacgacccgcgc acggcgcggt 2100 tcggtgatgc cacgatcctc gccctgctgg cgaagatcgaagagaagcag gacgagcttg 2160 gcaaggtcat gatgggcgtg gtccgcccga gggcagagccatgacttttt tagccgctaa 2220 aacggccggg gggtgcgcgt gattgccaag cacgtccccatgcgctccat caagaagagc 2280 gacttcgcgg agctggtgaa gtacatcacc gacgagcaaggcaagaccga gcgcctttgc 2340 gacgctcacc gggctggttg ccctcgccgc tgggctggcggccgtctatg gccctgcaaa 2400 cgcgccagaa acgccgtcga agccgtgtgc gagacaccgcggccgccggc gttgtggata 2460 cctcgcggaa aacttggccc tcactgacag atgaggggcggacgttgaca cttgaggggc 2520 cgactcaccc ggcgcggcgt tgacagatga ggggcaggctcgatttcggc cggcgacgtg 2580 gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattttacgcgagtt tcccacagat 2640 gatgtggaca agcctgggga taagtgccct gcggtattgacacttgaggg gcgcgactac 2700 tgacagatga ggggcgcgat ccttgacact tgaggggcagagtgctgaca gatgaggggc 2760 gcacctattg acatttgagg ggctgtccac aggcagaaaatccagcattt gcaagggttt 2820 ccgcccgttt ttcggccacc gctaacctgt cttttaacctgcttttaaac caatatttat 2880 aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtgaccgcgcacgc cgaagggggg 2940 tgccccccct tctcgaaccc tcccggcccg ctaacgcgggcctcccatcc ccccaggggc 3000 tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggcagcgctggca gtccttgcca 3060 ttgccgggat cggggcagta acgggatggg cgatcagcccgagcgcgacg cccggaagca 3120 ttgacgtgcc gcaggtgctg gcatcgacat tcagcgaccaggtgccgggc agtgagggcg 3180 gcggcctggg tggcggcctg cccttcactt cggccgtcggggcattcacg gacttcatgg 3240 cggggccggc aatttttacc ttgggcattc ttggcatagtggtcgcgggt gccgtgctcg 3300 tgttcggggg tgcgataaac ccagcgaacc atttgaggtgataggtaaga ttataccgag 3360 gtatgaaaac gagaattgga cctttacaga attactctatgaagcgccat atttaaaaag 3420 ctaccaagac gaagaggatg aagaggatga ggaggcagattgccttgaat atattgacaa 3480 tactgataag ataatatatc ttttatatag aagatatcgccgtatgtaag gatttcaggg 3540 ggcaaggcat aggcagcgcg cttatcaata tatctatagaatgggcaaag cataaaaact 3600 tgcatggact aatgcttgaa acccaggaca ataaccttatagcttgtaaa ttctatcata 3660 attgggtaat gactccaact tattgatagt gttttatgttcagataatgc ccgatgactt 3720 tgtcatgcag ctccaccgat tttgagaacg acagcgacttccgtcccagc cgtgccaggt 3780 gctgcctcag attcaggtta tgccgctcaa ttcgctgcgtatatcgcttg ctgattacgt 3840 gcagctttcc cttcaggcgg gattcataca gcggccagccatccgtcatc catatcacca 3900 cgtcaaaggg tgacagcagg ctcataagac gccccagcgtcgccatagtg cgttcaccga 3960 atacgtgcgc aacaaccgtc ttccggagac tgtcatacgcgtaaaacagc cagcgctggc 4020 gcgatttagc cccgacatag ccccactgtt cgtccatttccgcgcagacg atgacgtcac 4080 tgcccggctg tatgcgcgag gttaccgact gcggcctgagttttttaagt gacgtaaaat 4140 cgtgttgagg ccaacgccca taatgcgggc tgttgcccggcatccaacgc cattcatggc 4200 catatcaatg attttctggt gcgtaccggg ttgagaagcggtgtaagtga actgcagttg 4260 ccatgtttta cggcagtgag agcagagata gcgctgatgtccggcggtgc ttttgccgtt 4320 acgcaccacc ccgtcagtag ctgaacagga gggacagctgatagacacag aagccactgg 4380 agcacctcaa aaacaccatc atacactaaa tcagtaagttggcagcatca cccataattg 4440 tggtttcaaa atcggctccg tcgatactat gttatacgccaactttgaaa acaactttga 4500 aaaagctgtt ttctggtatt taaggtttta gaatgcaaggaacagtgaat tggagttcgt 4560 cttgttataa ttagcttctt ggggtatctt taaatactgtagaaaagagg aaggaaataa 4620 taaatggcta aaatgagaat atcaccggaa ttgaaaaaactgatcgaaaa ataccgctgc 4680 gtaaaagata cggaaggaat gtctcctgct aaggtatataagctggtggg agaaaatgaa 4740 aacctatatt taaaaatgac ggacagccgg tataaagggaccacctatga tgtggaacgg 4800 gaaaaggaca tgatgctatg gctggaagga aagctgcctgttccaaaggt cctgcacttt 4860 gaacggcatg atggctggag caatctgctc atgagtgaggccgatggcgt cctttgctcg 4920 gaagagtatg aagatgaaca aagccctgaa aagattatcgagctgtatgc ggagtgcatc 4980 aggctctttc actccatcga catatcggat tgtccctatacgaatagctt agacagccgc 5040 ttagccgaat tggattactt actgaataac gatctggccgatgtggattg cgaaaactgg 5100 gaagaagaca ctccatttaa agatccgcgc gagctgtatgattttttaaa gacggaaaag 5160 cccgaagagg aacttgtctt ttcccacggc gacctgggagacagcaacat ctttgtgaaa 5220 gatggcaaag taagtggctt tattgatctt gggagaagcggcagggcgga caagtggtat 5280 gacattgcct tctgcgtccg gtcgatcagg gaggatatcggggaagaaca gtatgtcgag 5340 ctattttttg acttactggg gatcaagcct gattgggagaaaataaaata ttatatttta 5400 ctggatgaat tgttttagta cctagatgtg gcgcaacgatgccggcgaca agcaggagcg 5460 caccgacttc ttccgcatca agtgttttgg ctctcaggccgaggcccacg gcaagtattt 5520 gggcaagggg tcgctggtat tcgtgcaggg caagattcggaataccaagt acgagaagga 5580 cggccagacg gtctacggga ccgacttcat tgccgataaggtggattatc tggacaccaa 5640 ggcaccaggc gggtcaaatc aggaataagg gcacattgccccggcgtgag tcggggcaat 5700 cccgcaagga gggtgaatga atcggacgtt tgaccggaaggcatacaggc aagaactgat 5760 cgacgcgggg ttttccgccg aggatgccga aaccatcgcaagccgcaccg tcatgcgtgc 5820 gccccgcgaa accttccagt ccgtcggctc gatggtccagcaagctacgg ccaagatcga 5880 gcgcgacagc gtgcaactgg ctccccctgc cctgcccgcgccatcggccg ccgtggagcg 5940 ttcgcgtcgt ctcgaacagg aggcggcagg tttggcgaagtcgatgacca tcgacacgcg 6000 aggaactatg acgaccaaga agcgaaaaac cgccggcgaggacctggcaa aacaggtcag 6060 cgaggccaag caggccgcgt tgctgaaaca cacgaagcagcagatcaagg aaatgcagct 6120 ttccttgttc gatattgcgc cgtggccgga cacgatgcgagcgatgccaa acgacacggc 6180 ccgctctgcc ctgttcacca cgcgcaacaa gaaaatcccgcgcgaggcgc tgcaaaacaa 6240 ggtcattttc cacgtcaaca aggacgtgaa gatcacctacaccggcgtcg agctgcgggc 6300 cgacgatgac gaactggtgt ggcagcaggt gttggagtacgcgaagcgca cccctatcgg 6360 cgagccgatc accttcacgt tctacgagct ttgccaggacctgggctggt cgatcaatgg 6420 ccggtattac acgaaggccg aggaatgcct gtcgcgcctacaggcgacgg cgatgggctt 6480 cacgtccgac cgcgttgggc acctggaatc ggtgtcgctgctgcaccgct tccgcgtcct 6540 ggaccgtggc aagaaaacgt cccgttgcca ggtcctgatcgacgaggaaa tcgtcgtgct 6600 gtttgctggc gaccactaca cgaaattcat atgggagaagtaccgcaagc tgtcgccgac 6660 ggcccgacgg atgttcgact atttcagctc gcaccgggagccgtacccgc tcaagctgga 6720 aaccttccgc ctcatgtgcg gatcggattc cacccgcgtgaagaagtggc gcgagcaggt 6780 cggcgaagcc tgcgaagagt tgcgaggcag cggcctggtggaacacgcct gggtcaatga 6840 tgacctggtg cattgcaaac gctagggcct tgtggggtcagttccggctg ggggttcagc 6900 agccagcgct ttactggcat ttcaggaaca agcgggcactgctcgacgca cttgcttcgc 6960 tcagtatcgc tcgggacgca cggcgcgctc tacgaactgccgataaacag aggattaaaa 7020 ttgacaattg tgattaaggc tcagattcga cggcttggagcggccgacgt gcaggatttc 7080 cgcgagatcc gattgtcggc cctgaagaaa gctccagagatgttcgggtc cgtttacgag 7140 cacgaggaga aaaagcccat ggaggcgttc gctgaacggttgcgagatgc cgtggcattc 7200 ggcgcctaca tcgacggcga gatcattggg ctgtcggtcttcaaacagga ggacggcccc 7260 aaggacgctc acaaggcgca tctgtccggc gttttcgtggagcccgaaca gcgaggccga 7320 ggggtcgccg gtatgctgct gcgggcgttg ccggcgggtttattgctcgt gatgatcgtc 7380 cgacagattc caacgggaat ctggtggatg cgcatcttcatcctcggcgc acttaatatt 7440 tcgctattct ggagcttgtt gtttatttcg gtctaccgcctgccgggcgg ggtcgcggcg 7500 acggtaggcg ctgtgcagcc gctgatggtc gtgttcatctctgccgctct gctaggtagc 7560 ccgatacgat tgatggcggt cctgggggct atttgcggaactgcgggcgt ggcgctgttg 7620 gtgttgacac caaacgcagc gctagatcct gtcggcgtcgcagcgggcct ggcgggggcg 7680 gtttccatgg cgttcggaac cgtgctgacc cgcaagtggcaacctcccgt gcctctgctc 7740 acctttaccg cctggcaact ggcggccgga ggacttctgctcgttccagt agctttagtg 7800 tttgatccgc caatcccgat gcctacagga accaatgttctcggcctggc gtggctcggc 7860 ctgatcggag cgggtttaac ctacttcctt tggttccgggggatctcgcg actcgaacct 7920 acagttgttt ccttactggg ctttctcagc cccagatctggggtcgatca gccggggatg 7980 catcaggccg acagtcggaa cttcgggtcc ccgacctgtaccattcggtg agcaatggat 8040 aggggagttg atatcgtcaa cgttcacttc taaagaaatagcgccactca gcttcctcag 8100 cggctttatc cagcgatttc ctattatgtc ggcatagttctcaagatcga cagcctgtca 8160 cggttaagcg agaaatgaat aagaaggctg ataattcggatctctgcgag ggagatgata 8220 tttgatcaca ggcagcaacg ctctgtcatc gttacaatcaacatgctacc ctccgcgaga 8280 tcatccgtgt ttcaaacccg gcagcttagt tgccgttcttccgaatagca tcggtaacat 8340 gagcaaagtc tgccgcctta caacggctct cccgctgacgccgtcccgga ctgatgggct 8400 gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcggggagctgtt ggctggctgg 8460 tggcaggata tattgtggtg taaacaaatt gacgcttagacaacttaata acacattgcg 8520 gacgttttta atgtactggg gtggtttttc ttttcaccagtgagacgggc aacagctgat 8580 tgcccttcac cgcctggccc tgagagagtt gcagcaagcggtccacgctg gtttgcccca 8640 gcaggcgaaa atcctgtttg atggtggttc cgaaatcggcaaaatccctt ataaatcaaa 8700 agaatagccc gagatagggt tgagtgttgt tccagtttggaacaagagtc cactattaaa 8760 gaacgtggac tccaacgtca aagggcgaaa aaccgtctatcagggcgatg gcccactacg 8820 tgaaccatca cccaaatcaa gttttttggg gtcgaggtgccgtaaagcac taaatcggaa 8880 ccctaaaggg agcccccgat ttagagcttg acggggaaagccggcgaacg tggcgagaaa 8940 ggaagggaag aaagcgaaag gagcgggcgc cattcaggctgcgcaactgt tgggaagggc 9000 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaaagggggatgt gctgcaaggc 9060 gattaagttg ggtaacgcca gggttttccc agtcacgacgttgtaaaacg acggccagtg 9120 aattaattcc catcttgaaa gaaatatagt ttaaatatttattgataaaa taacaagtca 9180 ggtattatag tccaagcaaa aacataaatt tattgatgcaagtttaaatt cagaaatatt 9240 tcaataactg attatatcag ctggtacatt gccgtagatgaaagactgag tgcgatatta 9300 tgtgtaatac ataaattgat gatatagcta gcttagctcatcgggggatc cgtcgaagct 9360 agcttgggtc ccgctcagaa gaactcgtca agaaggcgatagaaggcgat gcgctgcgaa 9420 tcgggagcgg cgataccgta aagcacgagg aagcggtcagcccattcgcc gccaagctct 9480 tcagcaatat cacgggtagc caacgctatg tcctgatagcggtccgccac acccagccgg 9540 ccacagtcga tgaatccaga aaagcggcca ttttccaccatgatattcgg caagcaggca 9600 tcgccatggg tcacgacgag atcctcgccg tcgggcatgcgcgccttgag cctggcgaac 9660 agttcggctg gcgcgagccc ctgatgctct tcgtccagatcatcctgatc gacaagaccg 9720 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcgcttggtggtc gaatgggcag 9780 gtagccggat caagcgtatg cagccgccgc attgcatcagccatgatgga tactttctcg 9840 gcaggagcaa ggtgagatga caggagatcc tgccccggcacttcgcccaa tagcagccag 9900 tcccttcccg cttcagtgac aacgtcgagc acagctgcgcaaggaacgcc cgtcgtggcc 9960 agccacgata gccgcgctgc ctcgtcctgc agttcattcagggcaccgga caggtcggtc 10020 ttgacaaaaa gaaccgggcg cccctgcgct gacagccggaacacggcggc atcagagcag 10080 ccgattgtct gttgtgccca gtcatagccg aatagcctctccacccaagc ggccggagaa 10140 cctgcgtgca atccatcttg ttcaatccaa gctcccatgggccctcgact agagtcgaga 10200 tctggattga gagtgaatat gagactctaa ttggataccgaggggaattt atggaacgtc 10260 agtggagcat ttttgacaag aaatatttgc tagctgatagtgaccttagg cgacttttga 10320 acgcgcaata atggtttctg acgtatgtgc ttagctcattaaactccaga aacccgcggc 10380 tgagtggctc cttcaacgtt gcggttctgt cagttccaaacgtaaaacgg cttgtcccgc 10440 gtcatcggcg ggggtcataa cgtgactccc ttaattctccgctcatgatc ttgatcccct 10500 gcgccatcag atccttggcg gcaagaaagc catccagtttactttgcagg gcttcccaac 10560 cttaccagag ggcgccccag ctggcaattc cggttcgcttgctgtccata aaaccgccca 10620 gtctagctat cgccatgtaa gcccactgca agctacctgctttctctttg cgcttgcgtt 10680 ttcccttgtc cagatagccc agtagctgac attcatccggggtcagcacc gtttctgcgg 10740 actggctttc tacgtgttcc gcttccttta gcagcccttgcgccctgagt gcttgcggca 10800 gcgtgaagct tgcatgcctg caggtcgacg gcgcgccgagctcctcgagc aaatttacac 10860 attgccacta aacgtctaaa cccttgtaat ttgtttttgttttactatgt gtgttatgta 10920 tttgatttgc gataaatttt tatatttggt actaaatttataacaccttt tatgctaacg 10980 tttgccaaca cttagcaatt tgcaagttga ttaattgattctaaattatt tttgtcttct 11040 aaatacatat actaatcaac tggaaatgta aatatttgctaatatttcta ctataggaga 11100 attaaagtga gtgaatatgg taccacaagg tttggagatttaattgttgc aatgctgcat 11160 ggatggcata tacaccaaac attcaataat tcttgaggataataatggta ccacacaaga 11220 tttgaggtgc atgaacgtca cgtggacaaa aggtttagtaatttttcaag acaacaatgt 11280 taccacacac aagttttgag gtgcatgcat ggatgccctgtggaaagttt aaaaatattt 11340 tggaaatgat ttgcatggaa gccatgtgta aaaccatgacatccacttgg aggatgcaat 11400 aatgaagaaa actacaaatt tacatgcaac tagttatgcatgtagtctat ataatgagga 11460 ttttgcaata ctttcattca tacacactca ctaagttttacacgattata atttcttcat 11520 agccagcgga tccgatatcg ggcccgctag cgttaaccctgctttaatga gatatgcgag 11580 acgcctatga tcgcatgata tttgctttca attctgttgtgcacgttgta aaaaacctga 11640 gcatgtgtag ctcagatcct taccgccggt ttcggttcattctaatgaat atatcacccg 11700 ttactatcgt atttttatga ataatattct ccgttcaatttactgattgt ccgtcgacga 11760 attcgagctc ggcgcgcctc tagaggatcg atgaattcagatcggctgag tggctccttc 11820 aacgttgcgg ttctgtcagt tccaaacgta aaacggcttgtcccgcgtca tcggcggggg 11880 tcataacgtg actcccttaa ttctccgctc atgatcagattgtcgtttcc cgccttcagt 11940 ttaaactatc agtgtttgac aggatatatt ggcgggtaaacctaagagaa aagagcgttt 12000 attagaataa tcggatattt aaaagggcgt gaaaaggtttatccttcgtc catttgtatg 12060 tgcatgccaa ccacagggtt cccca 12085 22 12079DNA Unknown Plant expression vector with a promoter- terminatorexpression cassette 22 gatctggcgc cggccagcga gacgagcaag attggccgccgcccgaaacg atccgacagc 60 gcgcccagca caggtgcgca ggcaaattgc accaacgcatacagcgccag cagaatgcca 120 tagtgggcgg tgacgtcgtt cgagtgaacc agatcgcgcaggaggcccgg cagcaccggc 180 ataatcaggc cgatgccgac agcgtcgagc gcgacagtgctcagaattac gatcaggggt 240 atgttgggtt tcacgtctgg cctccggacc agcctccgctggtccgattg aacgcgcgga 300 ttctttatca ctgataagtt ggtggacata ttatgtttatcagtgataaa gtgtcaagca 360 tgacaaagtt gcagccgaat acagtgatcc gtgccgccctggacctgttg aacgaggtcg 420 gcgtagacgg tctgacgaca cgcaaactgg cggaacggttgggggttcag cagccggcgc 480 tttactggca cttcaggaac aagcgggcgc tgctcgacgcactggccgaa gccatgctgg 540 cggagaatca tacgcattcg gtgccgagag ccgacgacgactggcgctca tttctgatcg 600 ggaatgcccg cagcttcagg caggcgctgc tcgcctaccgcgatggcgcg cgcatccatg 660 ccggcacgcg accgggcgca ccgcagatgg aaacggccgacgcgcagctt cgcttcctct 720 gcgaggcggg tttttcggcc ggggacgccg tcaatgcgctgatgacaatc agctacttca 780 ctgttggggc cgtgcttgag gagcaggccg gcgacagcgatgccggcgag cgcggcggca 840 ccgttgaaca ggctccgctc tcgccgctgt tgcgggccgcgatagacgcc ttcgacgaag 900 ccggtccgga cgcagcgttc gagcagggac tcgcggtgattgtcgatgga ttggcgaaaa 960 ggaggctcgt tgtcaggaac gttgaaggac cgagaaagggtgacgattga tcaggaccgc 1020 tgccggagcg caacccactc actacagcag agccatgtagacaacatccc ctcccccttt 1080 ccaccgcgtc agacgcccgt agcagcccgc tacgggctttttcatgccct gccctagcgt 1140 ccaagcctca cggccgcgct cggcctctct ggcggccttctggcgctctt ccgcttcctc 1200 gctcactgac tcgctgcgct cggtcgttcg gctgcggcgagcggtatcag ctcactcaaa 1260 ggcggtaata cggttatcca cagaatcagg ggataacgcaggaaagaaca tgtgagcaaa 1320 aggccagcaa aaggccagga accgtaaaaa ggccgcgttgctggcgtttt tccataggct 1380 ccgcccccct gacgagcatc acaaaaatcg acgctcaagtcagaggtggc gaaacccgac 1440 aggactataa agataccagg cgtttccccc tggaagctccctcgtgcgct ctcctgttcc 1500 gaccctgccg cttaccggat acctgtccgc ctttctcccttcgggaagcg tggcgctttt 1560 ccgctgcata accctgcttc ggggtcatta tagcgattttttcggtatat ccatcctttt 1620 tcgcacgata tacaggattt tgccaaaggg ttcgtgtagactttccttgg tgtatccaac 1680 ggcgtcagcc gggcaggata ggtgaagtag gcccacccgcgagcgggtgt tccttcttca 1740 ctgtccctta ttcgcacctg gcggtgctca acgggaatcctgctctgcga ggctggccgg 1800 ctaccgccgg cgtaacagat gagggcaagc ggatggctgatgaaaccaag ccaaccagga 1860 agggcagccc acctatcaag gtgtactgcc ttccagacgaacgaagagcg attgaggaaa 1920 aggcggcggc ggccggcatg agcctgtcgg cctacctgctggccgtcggc cagggctaca 1980 aaatcacggg cgtcgtggac tatgagcacg tccgcgagctggcccgcatc aatggcgacc 2040 tgggccgcct gggcggcctg ctgaaactct ggctcaccgacgacccgcgc acggcgcggt 2100 tcggtgatgc cacgatcctc gccctgctgg cgaagatcgaagagaagcag gacgagcttg 2160 gcaaggtcat gatgggcgtg gtccgcccga gggcagagccatgacttttt tagccgctaa 2220 aacggccggg gggtgcgcgt gattgccaag cacgtccccatgcgctccat caagaagagc 2280 gacttcgcgg agctggtgaa gtacatcacc gacgagcaaggcaagaccga gcgcctttgc 2340 gacgctcacc gggctggttg ccctcgccgc tgggctggcggccgtctatg gccctgcaaa 2400 cgcgccagaa acgccgtcga agccgtgtgc gagacaccgcggccgccggc gttgtggata 2460 cctcgcggaa aacttggccc tcactgacag atgaggggcggacgttgaca cttgaggggc 2520 cgactcaccc ggcgcggcgt tgacagatga ggggcaggctcgatttcggc cggcgacgtg 2580 gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattttacgcgagtt tcccacagat 2640 gatgtggaca agcctgggga taagtgccct gcggtattgacacttgaggg gcgcgactac 2700 tgacagatga ggggcgcgat ccttgacact tgaggggcagagtgctgaca gatgaggggc 2760 gcacctattg acatttgagg ggctgtccac aggcagaaaatccagcattt gcaagggttt 2820 ccgcccgttt ttcggccacc gctaacctgt cttttaacctgcttttaaac caatatttat 2880 aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtgaccgcgcacgc cgaagggggg 2940 tgccccccct tctcgaaccc tcccggcccg ctaacgcgggcctcccatcc ccccaggggc 3000 tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggcagcgctggca gtccttgcca 3060 ttgccgggat cggggcagta acgggatggg cgatcagcccgagcgcgacg cccggaagca 3120 ttgacgtgcc gcaggtgctg gcatcgacat tcagcgaccaggtgccgggc agtgagggcg 3180 gcggcctggg tggcggcctg cccttcactt cggccgtcggggcattcacg gacttcatgg 3240 cggggccggc aatttttacc ttgggcattc ttggcatagtggtcgcgggt gccgtgctcg 3300 tgttcggggg tgcgataaac ccagcgaacc atttgaggtgataggtaaga ttataccgag 3360 gtatgaaaac gagaattgga cctttacaga attactctatgaagcgccat atttaaaaag 3420 ctaccaagac gaagaggatg aagaggatga ggaggcagattgccttgaat atattgacaa 3480 tactgataag ataatatatc ttttatatag aagatatcgccgtatgtaag gatttcaggg 3540 ggcaaggcat aggcagcgcg cttatcaata tatctatagaatgggcaaag cataaaaact 3600 tgcatggact aatgcttgaa acccaggaca ataaccttatagcttgtaaa ttctatcata 3660 attgggtaat gactccaact tattgatagt gttttatgttcagataatgc ccgatgactt 3720 tgtcatgcag ctccaccgat tttgagaacg acagcgacttccgtcccagc cgtgccaggt 3780 gctgcctcag attcaggtta tgccgctcaa ttcgctgcgtatatcgcttg ctgattacgt 3840 gcagctttcc cttcaggcgg gattcataca gcggccagccatccgtcatc catatcacca 3900 cgtcaaaggg tgacagcagg ctcataagac gccccagcgtcgccatagtg cgttcaccga 3960 atacgtgcgc aacaaccgtc ttccggagac tgtcatacgcgtaaaacagc cagcgctggc 4020 gcgatttagc cccgacatag ccccactgtt cgtccatttccgcgcagacg atgacgtcac 4080 tgcccggctg tatgcgcgag gttaccgact gcggcctgagttttttaagt gacgtaaaat 4140 cgtgttgagg ccaacgccca taatgcgggc tgttgcccggcatccaacgc cattcatggc 4200 catatcaatg attttctggt gcgtaccggg ttgagaagcggtgtaagtga actgcagttg 4260 ccatgtttta cggcagtgag agcagagata gcgctgatgtccggcggtgc ttttgccgtt 4320 acgcaccacc ccgtcagtag ctgaacagga gggacagctgatagacacag aagccactgg 4380 agcacctcaa aaacaccatc atacactaaa tcagtaagttggcagcatca cccataattg 4440 tggtttcaaa atcggctccg tcgatactat gttatacgccaactttgaaa acaactttga 4500 aaaagctgtt ttctggtatt taaggtttta gaatgcaaggaacagtgaat tggagttcgt 4560 cttgttataa ttagcttctt ggggtatctt taaatactgtagaaaagagg aaggaaataa 4620 taaatggcta aaatgagaat atcaccggaa ttgaaaaaactgatcgaaaa ataccgctgc 4680 gtaaaagata cggaaggaat gtctcctgct aaggtatataagctggtggg agaaaatgaa 4740 aacctatatt taaaaatgac ggacagccgg tataaagggaccacctatga tgtggaacgg 4800 gaaaaggaca tgatgctatg gctggaagga aagctgcctgttccaaaggt cctgcacttt 4860 gaacggcatg atggctggag caatctgctc atgagtgaggccgatggcgt cctttgctcg 4920 gaagagtatg aagatgaaca aagccctgaa aagattatcgagctgtatgc ggagtgcatc 4980 aggctctttc actccatcga catatcggat tgtccctatacgaatagctt agacagccgc 5040 ttagccgaat tggattactt actgaataac gatctggccgatgtggattg cgaaaactgg 5100 gaagaagaca ctccatttaa agatccgcgc gagctgtatgattttttaaa gacggaaaag 5160 cccgaagagg aacttgtctt ttcccacggc gacctgggagacagcaacat ctttgtgaaa 5220 gatggcaaag taagtggctt tattgatctt gggagaagcggcagggcgga caagtggtat 5280 gacattgcct tctgcgtccg gtcgatcagg gaggatatcggggaagaaca gtatgtcgag 5340 ctattttttg acttactggg gatcaagcct gattgggagaaaataaaata ttatatttta 5400 ctggatgaat tgttttagta cctagatgtg gcgcaacgatgccggcgaca agcaggagcg 5460 caccgacttc ttccgcatca agtgttttgg ctctcaggccgaggcccacg gcaagtattt 5520 gggcaagggg tcgctggtat tcgtgcaggg caagattcggaataccaagt acgagaagga 5580 cggccagacg gtctacggga ccgacttcat tgccgataaggtggattatc tggacaccaa 5640 ggcaccaggc gggtcaaatc aggaataagg gcacattgccccggcgtgag tcggggcaat 5700 cccgcaagga gggtgaatga atcggacgtt tgaccggaaggcatacaggc aagaactgat 5760 cgacgcgggg ttttccgccg aggatgccga aaccatcgcaagccgcaccg tcatgcgtgc 5820 gccccgcgaa accttccagt ccgtcggctc gatggtccagcaagctacgg ccaagatcga 5880 gcgcgacagc gtgcaactgg ctccccctgc cctgcccgcgccatcggccg ccgtggagcg 5940 ttcgcgtcgt ctcgaacagg aggcggcagg tttggcgaagtcgatgacca tcgacacgcg 6000 aggaactatg acgaccaaga agcgaaaaac cgccggcgaggacctggcaa aacaggtcag 6060 cgaggccaag caggccgcgt tgctgaaaca cacgaagcagcagatcaagg aaatgcagct 6120 ttccttgttc gatattgcgc cgtggccgga cacgatgcgagcgatgccaa acgacacggc 6180 ccgctctgcc ctgttcacca cgcgcaacaa gaaaatcccgcgcgaggcgc tgcaaaacaa 6240 ggtcattttc cacgtcaaca aggacgtgaa gatcacctacaccggcgtcg agctgcgggc 6300 cgacgatgac gaactggtgt ggcagcaggt gttggagtacgcgaagcgca cccctatcgg 6360 cgagccgatc accttcacgt tctacgagct ttgccaggacctgggctggt cgatcaatgg 6420 ccggtattac acgaaggccg aggaatgcct gtcgcgcctacaggcgacgg cgatgggctt 6480 cacgtccgac cgcgttgggc acctggaatc ggtgtcgctgctgcaccgct tccgcgtcct 6540 ggaccgtggc aagaaaacgt cccgttgcca ggtcctgatcgacgaggaaa tcgtcgtgct 6600 gtttgctggc gaccactaca cgaaattcat atgggagaagtaccgcaagc tgtcgccgac 6660 ggcccgacgg atgttcgact atttcagctc gcaccgggagccgtacccgc tcaagctgga 6720 aaccttccgc ctcatgtgcg gatcggattc cacccgcgtgaagaagtggc gcgagcaggt 6780 cggcgaagcc tgcgaagagt tgcgaggcag cggcctggtggaacacgcct gggtcaatga 6840 tgacctggtg cattgcaaac gctagggcct tgtggggtcagttccggctg ggggttcagc 6900 agccagcgct ttactggcat ttcaggaaca agcgggcactgctcgacgca cttgcttcgc 6960 tcagtatcgc tcgggacgca cggcgcgctc tacgaactgccgataaacag aggattaaaa 7020 ttgacaattg tgattaaggc tcagattcga cggcttggagcggccgacgt gcaggatttc 7080 cgcgagatcc gattgtcggc cctgaagaaa gctccagagatgttcgggtc cgtttacgag 7140 cacgaggaga aaaagcccat ggaggcgttc gctgaacggttgcgagatgc cgtggcattc 7200 ggcgcctaca tcgacggcga gatcattggg ctgtcggtcttcaaacagga ggacggcccc 7260 aaggacgctc acaaggcgca tctgtccggc gttttcgtggagcccgaaca gcgaggccga 7320 ggggtcgccg gtatgctgct gcgggcgttg ccggcgggtttattgctcgt gatgatcgtc 7380 cgacagattc caacgggaat ctggtggatg cgcatcttcatcctcggcgc acttaatatt 7440 tcgctattct ggagcttgtt gtttatttcg gtctaccgcctgccgggcgg ggtcgcggcg 7500 acggtaggcg ctgtgcagcc gctgatggtc gtgttcatctctgccgctct gctaggtagc 7560 ccgatacgat tgatggcggt cctgggggct atttgcggaactgcgggcgt ggcgctgttg 7620 gtgttgacac caaacgcagc gctagatcct gtcggcgtcgcagcgggcct ggcgggggcg 7680 gtttccatgg cgttcggaac cgtgctgacc cgcaagtggcaacctcccgt gcctctgctc 7740 acctttaccg cctggcaact ggcggccgga ggacttctgctcgttccagt agctttagtg 7800 tttgatccgc caatcccgat gcctacagga accaatgttctcggcctggc gtggctcggc 7860 ctgatcggag cgggtttaac ctacttcctt tggttccgggggatctcgcg actcgaacct 7920 acagttgttt ccttactggg ctttctcagc cccagatctggggtcgatca gccggggatg 7980 catcaggccg acagtcggaa cttcgggtcc ccgacctgtaccattcggtg agcaatggat 8040 aggggagttg atatcgtcaa cgttcacttc taaagaaatagcgccactca gcttcctcag 8100 cggctttatc cagcgatttc ctattatgtc ggcatagttctcaagatcga cagcctgtca 8160 cggttaagcg agaaatgaat aagaaggctg ataattcggatctctgcgag ggagatgata 8220 tttgatcaca ggcagcaacg ctctgtcatc gttacaatcaacatgctacc ctccgcgaga 8280 tcatccgtgt ttcaaacccg gcagcttagt tgccgttcttccgaatagca tcggtaacat 8340 gagcaaagtc tgccgcctta caacggctct cccgctgacgccgtcccgga ctgatgggct 8400 gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcggggagctgtt ggctggctgg 8460 tggcaggata tattgtggtg taaacaaatt gacgcttagacaacttaata acacattgcg 8520 gacgttttta atgtactggg gtggtttttc ttttcaccagtgagacgggc aacagctgat 8580 tgcccttcac cgcctggccc tgagagagtt gcagcaagcggtccacgctg gtttgcccca 8640 gcaggcgaaa atcctgtttg atggtggttc cgaaatcggcaaaatccctt ataaatcaaa 8700 agaatagccc gagatagggt tgagtgttgt tccagtttggaacaagagtc cactattaaa 8760 gaacgtggac tccaacgtca aagggcgaaa aaccgtctatcagggcgatg gcccactacg 8820 tgaaccatca cccaaatcaa gttttttggg gtcgaggtgccgtaaagcac taaatcggaa 8880 ccctaaaggg agcccccgat ttagagcttg acggggaaagccggcgaacg tggcgagaaa 8940 ggaagggaag aaagcgaaag gagcgggcgc cattcaggctgcgcaactgt tgggaagggc 9000 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaaagggggatgt gctgcaaggc 9060 gattaagttg ggtaacgcca gggttttccc agtcacgacgttgtaaaacg acggccagtg 9120 aattaattcc catcttgaaa gaaatatagt ttaaatatttattgataaaa taacaagtca 9180 ggtattatag tccaagcaaa aacataaatt tattgatgcaagtttaaatt cagaaatatt 9240 tcaataactg attatatcag ctggtacatt gccgtagatgaaagactgag tgcgatatta 9300 tgtgtaatac ataaattgat gatatagcta gcttagctcatcgggggatc cgtcgaagct 9360 agcttgggtc ccgctcagaa gaactcgtca agaaggcgatagaaggcgat gcgctgcgaa 9420 tcgggagcgg cgataccgta aagcacgagg aagcggtcagcccattcgcc gccaagctct 9480 tcagcaatat cacgggtagc caacgctatg tcctgatagcggtccgccac acccagccgg 9540 ccacagtcga tgaatccaga aaagcggcca ttttccaccatgatattcgg caagcaggca 9600 tcgccatggg tcacgacgag atcctcgccg tcgggcatgcgcgccttgag cctggcgaac 9660 agttcggctg gcgcgagccc ctgatgctct tcgtccagatcatcctgatc gacaagaccg 9720 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcgcttggtggtc gaatgggcag 9780 gtagccggat caagcgtatg cagccgccgc attgcatcagccatgatgga tactttctcg 9840 gcaggagcaa ggtgagatga caggagatcc tgccccggcacttcgcccaa tagcagccag 9900 tcccttcccg cttcagtgac aacgtcgagc acagctgcgcaaggaacgcc cgtcgtggcc 9960 agccacgata gccgcgctgc ctcgtcctgc agttcattcagggcaccgga caggtcggtc 10020 ttgacaaaaa gaaccgggcg cccctgcgct gacagccggaacacggcggc atcagagcag 10080 ccgattgtct gttgtgccca gtcatagccg aatagcctctccacccaagc ggccggagaa 10140 cctgcgtgca atccatcttg ttcaatccaa gctcccatgggccctcgact agagtcgaga 10200 tctggattga gagtgaatat gagactctaa ttggataccgaggggaattt atggaacgtc 10260 agtggagcat ttttgacaag aaatatttgc tagctgatagtgaccttagg cgacttttga 10320 acgcgcaata atggtttctg acgtatgtgc ttagctcattaaactccaga aacccgcggc 10380 tgagtggctc cttcaacgtt gcggttctgt cagttccaaacgtaaaacgg cttgtcccgc 10440 gtcatcggcg ggggtcataa cgtgactccc ttaattctccgctcatgatc ttgatcccct 10500 gcgccatcag atccttggcg gcaagaaagc catccagtttactttgcagg gcttcccaac 10560 cttaccagag ggcgccccag ctggcaattc cggttcgcttgctgtccata aaaccgccca 10620 gtctagctat cgccatgtaa gcccactgca agctacctgctttctctttg cgcttgcgtt 10680 ttcccttgtc cagatagccc agtagctgac attcatccggggtcagcacc gtttctgcgg 10740 actggctttc tacgtgttcc gcttccttta gcagcccttgcgccctgagt gcttgcggca 10800 gcgtgaagct tgcatgcctg caggtcgacg gcgcgccgagctcctcgagc aaatttacac 10860 attgccacta aacgtctaaa cccttgtaat ttgtttttgttttactatgt gtgttatgta 10920 tttgatttgc gataaatttt tatatttggt actaaatttataacaccttt tatgctaacg 10980 tttgccaaca cttagcaatt tgcaagttga ttaattgattctaaattatt tttgtcttct 11040 aaatacatat actaatcaac tggaaatgta aatatttgctaatatttcta ctataggaga 11100 attaaagtga gtgaatatgg taccacaagg tttggagatttaattgttgc aatgctgcat 11160 ggatggcata tacaccaaac attcaataat tcttgaggataataatggta ccacacaaga 11220 tttgaggtgc atgaacgtca cgtggacaaa aggtttagtaatttttcaag acaacaatgt 11280 taccacacac aagttttgag gtgcatgcat ggatgccctgtggaaagttt aaaaatattt 11340 tggaaatgat ttgcatggaa gccatgtgta aaaccatgacatccacttgg aggatgcaat 11400 aatgaagaaa actacaaatt tacatgcaac tagttatgcatgtagtctat ataatgagga 11460 ttttgcaata ctttcattca tacacactca ctaagttttacacgattata atttcttcat 11520 agccagcaga tctgccggca tcgatcccgg gccatggcctgctttaatga gatatgcgag 11580 acgcctatga tcgcatgata tttgctttca attctgttgtgcacgttgta aaaaacctga 11640 gcatgtgtag ctcagatcct taccgccggt ttcggttcattctaatgaat atatcacccg 11700 ttactatcgt atttttatga ataatattct ccgttcaatttactgattgt ccgtcgacga 11760 gctcggcgcg cctctagagg atcgatgaat tcagatcggctgagtggctc cttcaacgtt 11820 gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgcgtcatcggcg ggggtcataa 11880 cgtgactccc ttaattctcc gctcatgatc agattgtcgtttcccgcctt cagtttaaac 11940 tatcagtgtt tgacaggata tattggcggg taaacctaagagaaaagagc gtttattaga 12000 ataatcggat atttaaaagg gcgtgaaaag gtttatccttcgtccatttg tatgtgcatg 12060 ccaaccacag ggttcccca 12079 23 13002 DNAUnknown Plant expression vector with two promoter- terminator expressioncassettes 23 gatctggcgc cggccagcga gacgagcaag attggccgcc gcccgaaacgatccgacagc 60 gcgcccagca caggtgcgca ggcaaattgc accaacgcat acagcgccagcagaatgcca 120 tagtgggcgg tgacgtcgtt cgagtgaacc agatcgcgca ggaggcccggcagcaccggc 180 ataatcaggc cgatgccgac agcgtcgagc gcgacagtgc tcagaattacgatcaggggt 240 atgttgggtt tcacgtctgg cctccggacc agcctccgct ggtccgattgaacgcgcgga 300 ttctttatca ctgataagtt ggtggacata ttatgtttat cagtgataaagtgtcaagca 360 tgacaaagtt gcagccgaat acagtgatcc gtgccgccct ggacctgttgaacgaggtcg 420 gcgtagacgg tctgacgaca cgcaaactgg cggaacggtt gggggttcagcagccggcgc 480 tttactggca cttcaggaac aagcgggcgc tgctcgacgc actggccgaagccatgctgg 540 cggagaatca tacgcattcg gtgccgagag ccgacgacga ctggcgctcatttctgatcg 600 ggaatgcccg cagcttcagg caggcgctgc tcgcctaccg cgatggcgcgcgcatccatg 660 ccggcacgcg accgggcgca ccgcagatgg aaacggccga cgcgcagcttcgcttcctct 720 gcgaggcggg tttttcggcc ggggacgccg tcaatgcgct gatgacaatcagctacttca 780 ctgttggggc cgtgcttgag gagcaggccg gcgacagcga tgccggcgagcgcggcggca 840 ccgttgaaca ggctccgctc tcgccgctgt tgcgggccgc gatagacgccttcgacgaag 900 ccggtccgga cgcagcgttc gagcagggac tcgcggtgat tgtcgatggattggcgaaaa 960 ggaggctcgt tgtcaggaac gttgaaggac cgagaaaggg tgacgattgatcaggaccgc 1020 tgccggagcg caacccactc actacagcag agccatgtag acaacatcccctcccccttt 1080 ccaccgcgtc agacgcccgt agcagcccgc tacgggcttt ttcatgccctgccctagcgt 1140 ccaagcctca cggccgcgct cggcctctct ggcggccttc tggcgctcttccgcttcctc 1200 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcagctcactcaaa 1260 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaacatgtgagcaaa 1320 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttttccataggct 1380 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggcgaaacccgac 1440 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgctctcctgttcc 1500 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcgtggcgctttt 1560 ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatatccatcctttt 1620 tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttggtgtatccaac 1680 ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgttccttcttca 1740 ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcgaggctggccgg 1800 ctaccgccgg cgtaacagat gagggcaagc ggatggctga tgaaaccaagccaaccagga 1860 agggcagccc acctatcaag gtgtactgcc ttccagacga acgaagagcgattgaggaaa 1920 aggcggcggc ggccggcatg agcctgtcgg cctacctgct ggccgtcggccagggctaca 1980 aaatcacggg cgtcgtggac tatgagcacg tccgcgagct ggcccgcatcaatggcgacc 2040 tgggccgcct gggcggcctg ctgaaactct ggctcaccga cgacccgcgcacggcgcggt 2100 tcggtgatgc cacgatcctc gccctgctgg cgaagatcga agagaagcaggacgagcttg 2160 gcaaggtcat gatgggcgtg gtccgcccga gggcagagcc atgacttttttagccgctaa 2220 aacggccggg gggtgcgcgt gattgccaag cacgtcccca tgcgctccatcaagaagagc 2280 gacttcgcgg agctggtgaa gtacatcacc gacgagcaag gcaagaccgagcgcctttgc 2340 gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatggccctgcaaa 2400 cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggcgttgtggata 2460 cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgacacttgaggggc 2520 cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggccggcgacgtg 2580 gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtttcccacagat 2640 gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggggcgcgactac 2700 tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgacagatgaggggc 2760 gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcatttgcaagggttt 2820 ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaaccaatatttat 2880 aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgccgaagggggg 2940 tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatccccccaggggc 3000 tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agcgctggcagtccttgcca 3060 ttgccgggat cggggcagta acgggatggg cgatcagccc gagcgcgacgcccggaagca 3120 ttgacgtgcc gcaggtgctg gcatcgacat tcagcgacca ggtgccgggcagtgagggcg 3180 gcggcctggg tggcggcctg cccttcactt cggccgtcgg ggcattcacggacttcatgg 3240 cggggccggc aatttttacc ttgggcattc ttggcatagt ggtcgcgggtgccgtgctcg 3300 tgttcggggg tgcgataaac ccagcgaacc atttgaggtg ataggtaagattataccgag 3360 gtatgaaaac gagaattgga cctttacaga attactctat gaagcgccatatttaaaaag 3420 ctaccaagac gaagaggatg aagaggatga ggaggcagat tgccttgaatatattgacaa 3480 tactgataag ataatatatc ttttatatag aagatatcgc cgtatgtaaggatttcaggg 3540 ggcaaggcat aggcagcgcg cttatcaata tatctataga atgggcaaagcataaaaact 3600 tgcatggact aatgcttgaa acccaggaca ataaccttat agcttgtaaattctatcata 3660 attgggtaat gactccaact tattgatagt gttttatgtt cagataatgcccgatgactt 3720 tgtcatgcag ctccaccgat tttgagaacg acagcgactt ccgtcccagccgtgccaggt 3780 gctgcctcag attcaggtta tgccgctcaa ttcgctgcgt atatcgcttgctgattacgt 3840 gcagctttcc cttcaggcgg gattcataca gcggccagcc atccgtcatccatatcacca 3900 cgtcaaaggg tgacagcagg ctcataagac gccccagcgt cgccatagtgcgttcaccga 3960 atacgtgcgc aacaaccgtc ttccggagac tgtcatacgc gtaaaacagccagcgctggc 4020 gcgatttagc cccgacatag ccccactgtt cgtccatttc cgcgcagacgatgacgtcac 4080 tgcccggctg tatgcgcgag gttaccgact gcggcctgag ttttttaagtgacgtaaaat 4140 cgtgttgagg ccaacgccca taatgcgggc tgttgcccgg catccaacgccattcatggc 4200 catatcaatg attttctggt gcgtaccggg ttgagaagcg gtgtaagtgaactgcagttg 4260 ccatgtttta cggcagtgag agcagagata gcgctgatgt ccggcggtgcttttgccgtt 4320 acgcaccacc ccgtcagtag ctgaacagga gggacagctg atagacacagaagccactgg 4380 agcacctcaa aaacaccatc atacactaaa tcagtaagtt ggcagcatcacccataattg 4440 tggtttcaaa atcggctccg tcgatactat gttatacgcc aactttgaaaacaactttga 4500 aaaagctgtt ttctggtatt taaggtttta gaatgcaagg aacagtgaattggagttcgt 4560 cttgttataa ttagcttctt ggggtatctt taaatactgt agaaaagaggaaggaaataa 4620 taaatggcta aaatgagaat atcaccggaa ttgaaaaaac tgatcgaaaaataccgctgc 4680 gtaaaagata cggaaggaat gtctcctgct aaggtatata agctggtgggagaaaatgaa 4740 aacctatatt taaaaatgac ggacagccgg tataaaggga ccacctatgatgtggaacgg 4800 gaaaaggaca tgatgctatg gctggaagga aagctgcctg ttccaaaggtcctgcacttt 4860 gaacggcatg atggctggag caatctgctc atgagtgagg ccgatggcgtcctttgctcg 4920 gaagagtatg aagatgaaca aagccctgaa aagattatcg agctgtatgcggagtgcatc 4980 aggctctttc actccatcga catatcggat tgtccctata cgaatagcttagacagccgc 5040 ttagccgaat tggattactt actgaataac gatctggccg atgtggattgcgaaaactgg 5100 gaagaagaca ctccatttaa agatccgcgc gagctgtatg attttttaaagacggaaaag 5160 cccgaagagg aacttgtctt ttcccacggc gacctgggag acagcaacatctttgtgaaa 5220 gatggcaaag taagtggctt tattgatctt gggagaagcg gcagggcggacaagtggtat 5280 gacattgcct tctgcgtccg gtcgatcagg gaggatatcg gggaagaacagtatgtcgag 5340 ctattttttg acttactggg gatcaagcct gattgggaga aaataaaatattatatttta 5400 ctggatgaat tgttttagta cctagatgtg gcgcaacgat gccggcgacaagcaggagcg 5460 caccgacttc ttccgcatca agtgttttgg ctctcaggcc gaggcccacggcaagtattt 5520 gggcaagggg tcgctggtat tcgtgcaggg caagattcgg aataccaagtacgagaagga 5580 cggccagacg gtctacggga ccgacttcat tgccgataag gtggattatctggacaccaa 5640 ggcaccaggc gggtcaaatc aggaataagg gcacattgcc ccggcgtgagtcggggcaat 5700 cccgcaagga gggtgaatga atcggacgtt tgaccggaag gcatacaggcaagaactgat 5760 cgacgcgggg ttttccgccg aggatgccga aaccatcgca agccgcaccgtcatgcgtgc 5820 gccccgcgaa accttccagt ccgtcggctc gatggtccag caagctacggccaagatcga 5880 gcgcgacagc gtgcaactgg ctccccctgc cctgcccgcg ccatcggccgccgtggagcg 5940 ttcgcgtcgt ctcgaacagg aggcggcagg tttggcgaag tcgatgaccatcgacacgcg 6000 aggaactatg acgaccaaga agcgaaaaac cgccggcgag gacctggcaaaacaggtcag 6060 cgaggccaag caggccgcgt tgctgaaaca cacgaagcag cagatcaaggaaatgcagct 6120 ttccttgttc gatattgcgc cgtggccgga cacgatgcga gcgatgccaaacgacacggc 6180 ccgctctgcc ctgttcacca cgcgcaacaa gaaaatcccg cgcgaggcgctgcaaaacaa 6240 ggtcattttc cacgtcaaca aggacgtgaa gatcacctac accggcgtcgagctgcgggc 6300 cgacgatgac gaactggtgt ggcagcaggt gttggagtac gcgaagcgcacccctatcgg 6360 cgagccgatc accttcacgt tctacgagct ttgccaggac ctgggctggtcgatcaatgg 6420 ccggtattac acgaaggccg aggaatgcct gtcgcgccta caggcgacggcgatgggctt 6480 cacgtccgac cgcgttgggc acctggaatc ggtgtcgctg ctgcaccgcttccgcgtcct 6540 ggaccgtggc aagaaaacgt cccgttgcca ggtcctgatc gacgaggaaatcgtcgtgct 6600 gtttgctggc gaccactaca cgaaattcat atgggagaag taccgcaagctgtcgccgac 6660 ggcccgacgg atgttcgact atttcagctc gcaccgggag ccgtacccgctcaagctgga 6720 aaccttccgc ctcatgtgcg gatcggattc cacccgcgtg aagaagtggcgcgagcaggt 6780 cggcgaagcc tgcgaagagt tgcgaggcag cggcctggtg gaacacgcctgggtcaatga 6840 tgacctggtg cattgcaaac gctagggcct tgtggggtca gttccggctgggggttcagc 6900 agccagcgct ttactggcat ttcaggaaca agcgggcact gctcgacgcacttgcttcgc 6960 tcagtatcgc tcgggacgca cggcgcgctc tacgaactgc cgataaacagaggattaaaa 7020 ttgacaattg tgattaaggc tcagattcga cggcttggag cggccgacgtgcaggatttc 7080 cgcgagatcc gattgtcggc cctgaagaaa gctccagaga tgttcgggtccgtttacgag 7140 cacgaggaga aaaagcccat ggaggcgttc gctgaacggt tgcgagatgccgtggcattc 7200 ggcgcctaca tcgacggcga gatcattggg ctgtcggtct tcaaacaggaggacggcccc 7260 aaggacgctc acaaggcgca tctgtccggc gttttcgtgg agcccgaacagcgaggccga 7320 ggggtcgccg gtatgctgct gcgggcgttg ccggcgggtt tattgctcgtgatgatcgtc 7380 cgacagattc caacgggaat ctggtggatg cgcatcttca tcctcggcgcacttaatatt 7440 tcgctattct ggagcttgtt gtttatttcg gtctaccgcc tgccgggcggggtcgcggcg 7500 acggtaggcg ctgtgcagcc gctgatggtc gtgttcatct ctgccgctctgctaggtagc 7560 ccgatacgat tgatggcggt cctgggggct atttgcggaa ctgcgggcgtggcgctgttg 7620 gtgttgacac caaacgcagc gctagatcct gtcggcgtcg cagcgggcctggcgggggcg 7680 gtttccatgg cgttcggaac cgtgctgacc cgcaagtggc aacctcccgtgcctctgctc 7740 acctttaccg cctggcaact ggcggccgga ggacttctgc tcgttccagtagctttagtg 7800 tttgatccgc caatcccgat gcctacagga accaatgttc tcggcctggcgtggctcggc 7860 ctgatcggag cgggtttaac ctacttcctt tggttccggg ggatctcgcgactcgaacct 7920 acagttgttt ccttactggg ctttctcagc cccagatctg gggtcgatcagccggggatg 7980 catcaggccg acagtcggaa cttcgggtcc ccgacctgta ccattcggtgagcaatggat 8040 aggggagttg atatcgtcaa cgttcacttc taaagaaata gcgccactcagcttcctcag 8100 cggctttatc cagcgatttc ctattatgtc ggcatagttc tcaagatcgacagcctgtca 8160 cggttaagcg agaaatgaat aagaaggctg ataattcgga tctctgcgagggagatgata 8220 tttgatcaca ggcagcaacg ctctgtcatc gttacaatca acatgctaccctccgcgaga 8280 tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt ccgaatagcatcggtaacat 8340 gagcaaagtc tgccgcctta caacggctct cccgctgacg ccgtcccggactgatgggct 8400 gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg gggagctgttggctggctgg 8460 tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaataacacattgcg 8520 gacgttttta atgtactggg gtggtttttc ttttcaccag tgagacgggcaacagctgat 8580 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctggtttgcccca 8640 gcaggcgaaa atcctgtttg atggtggttc cgaaatcggc aaaatcccttataaatcaaa 8700 agaatagccc gagatagggt tgagtgttgt tccagtttgg aacaagagtccactattaaa 8760 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatggcccactacg 8820 tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcactaaatcggaa 8880 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacgtggcgagaaa 8940 ggaagggaag aaagcgaaag gagcgggcgc cattcaggct gcgcaactgttgggaagggc 9000 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgtgctgcaaggc 9060 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacgacggccagtg 9120 aattaattcc catcttgaaa gaaatatagt ttaaatattt attgataaaataacaagtca 9180 ggtattatag tccaagcaaa aacataaatt tattgatgca agtttaaattcagaaatatt 9240 tcaataactg attatatcag ctggtacatt gccgtagatg aaagactgagtgcgatatta 9300 tgtgtaatac ataaattgat gatatagcta gcttagctca tcgggggatccgtcgaagct 9360 agcttgggtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgatgcgctgcgaa 9420 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgccgccaagctct 9480 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccacacccagccgg 9540 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcggcaagcaggca 9600 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgagcctggcgaac 9660 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatcgacaagaccg 9720 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtcgaatgggcag 9780 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatggatactttctcg 9840 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaatagcagccag 9900 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcccgtcgtggcc 9960 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccggacaggtcggtc 10020 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggcatcagagcag 10080 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagcggccggagaa 10140 cctgcgtgca atccatcttg ttcaatccaa gctcccatgg gccctcgactagagtcgaga 10200 tctggattga gagtgaatat gagactctaa ttggataccg aggggaatttatggaacgtc 10260 agtggagcat ttttgacaag aaatatttgc tagctgatag tgaccttaggcgacttttga 10320 acgcgcaata atggtttctg acgtatgtgc ttagctcatt aaactccagaaacccgcggc 10380 tgagtggctc cttcaacgtt gcggttctgt cagttccaaa cgtaaaacggcttgtcccgc 10440 gtcatcggcg ggggtcataa cgtgactccc ttaattctcc gctcatgatcttgatcccct 10500 gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagggcttcccaac 10560 cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccataaaaccgccca 10620 gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttgcgcttgcgtt 10680 ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcaccgtttctgcgg 10740 actggctttc tacgtgttcc gcttccttta gcagcccttg cgccctgagtgcttgcggca 10800 gcgtgaagct tgcatgcctg caggtcgacg gcgcgccgag ctcctcgagcaaatttacac 10860 attgccacta aacgtctaaa cccttgtaat ttgtttttgt tttactatgtgtgttatgta 10920 tttgatttgc gataaatttt tatatttggt actaaattta taacaccttttatgctaacg 10980 tttgccaaca cttagcaatt tgcaagttga ttaattgatt ctaaattatttttgtcttct 11040 aaatacatat actaatcaac tggaaatgta aatatttgct aatatttctactataggaga 11100 attaaagtga gtgaatatgg taccacaagg tttggagatt taattgttgcaatgctgcat 11160 ggatggcata tacaccaaac attcaataat tcttgaggat aataatggtaccacacaaga 11220 tttgaggtgc atgaacgtca cgtggacaaa aggtttagta atttttcaagacaacaatgt 11280 taccacacac aagttttgag gtgcatgcat ggatgccctg tggaaagtttaaaaatattt 11340 tggaaatgat ttgcatggaa gccatgtgta aaaccatgac atccacttggaggatgcaat 11400 aatgaagaaa actacaaatt tacatgcaac tagttatgca tgtagtctatataatgagga 11460 ttttgcaata ctttcattca tacacactca ctaagtttta cacgattataatttcttcat 11520 agccagccca ccgcggtggg cggccgcctg cagtctagaa ggcctcctgctttaatgaga 11580 tatgcgagac gcctatgatc gcatgatatt tgctttcaat tctgttgtgcacgttgtaaa 11640 aaacctgagc atgtgtagct cagatcctta ccgccggttt cggttcattctaatgaatat 11700 atcacccgtt actatcgtat ttttatgaat aatattctcc gttcaatttactgattgtcc 11760 gtcgagcaaa tttacacatt gccactaaac gtctaaaccc ttgtaatttgtttttgtttt 11820 actatgtgtg ttatgtattt gatttgcgat aaatttttat atttggtactaaatttataa 11880 caccttttat gctaacgttt gccaacactt agcaatttgc aagttgattaattgattcta 11940 aattattttt gtcttctaaa tacatatact aatcaactgg aaatgtaaatatttgctaat 12000 atttctacta taggagaatt aaagtgagtg aatatggtac cacaaggtttggagatttaa 12060 ttgttgcaat gctgcatgga tggcatatac accaaacatt caataattcttgaggataat 12120 aatggtacca cacaagattt gaggtgcatg aacgtcacgt ggacaaaaggtttagtaatt 12180 tttcaagaca acaatgttac cacacacaag ttttgaggtg catgcatggatgccctgtgg 12240 aaagtttaaa aatattttgg aaatgatttg catggaagcc atgtgtaaaaccatgacatc 12300 cacttggagg atgcaataat gaagaaaact acaaatttac atgcaactagttatgcatgt 12360 agtctatata atgaggattt tgcaatactt tcattcatac acactcactaagttttacac 12420 gattataatt tcttcatagc cagcggatcc gatatcgggc ccgctagcgttaaccctgct 12480 ttaatgagat atgcgagacg cctatgatcg catgatattt gctttcaattctgttgtgca 12540 cgttgtaaaa aacctgagca tgtgtagctc agatccttac cgccggtttcggttcattct 12600 aatgaatata tcacccgtta ctatcgtatt tttatgaata atattctccgttcaatttac 12660 tgattgtccg tcgacgaatt cgagctcggc gcgcctctag aggatcgatgaattcagatc 12720 ggctgagtgg ctccttcaac gttgcggttc tgtcagttcc aaacgtaaaacggcttgtcc 12780 cgcgtcatcg gcgggggtca taacgtgact cccttaattc tccgctcatgatcagattgt 12840 cgtttcccgc cttcagttta aactatcagt gtttgacagg atatattggcgggtaaacct 12900 aagagaaaag agcgtttatt agaataatcg gatatttaaa agggcgtgaaaaggtttatc 12960 cttcgtccat ttgtatgtgc atgccaacca cagggttccc ca 13002 2413905 DNA Unknown Plant expression vector with three promoter-terminator expression cassettes 24 gatctggcgc cggccagcga gacgagcaagattggccgcc gcccgaaacg atccgacagc 60 gcgcccagca caggtgcgca ggcaaattgcaccaacgcat acagcgccag cagaatgcca 120 tagtgggcgg tgacgtcgtt cgagtgaaccagatcgcgca ggaggcccgg cagcaccggc 180 ataatcaggc cgatgccgac agcgtcgagcgcgacagtgc tcagaattac gatcaggggt 240 atgttgggtt tcacgtctgg cctccggaccagcctccgct ggtccgattg aacgcgcgga 300 ttctttatca ctgataagtt ggtggacatattatgtttat cagtgataaa gtgtcaagca 360 tgacaaagtt gcagccgaat acagtgatccgtgccgccct ggacctgttg aacgaggtcg 420 gcgtagacgg tctgacgaca cgcaaactggcggaacggtt gggggttcag cagccggcgc 480 tttactggca cttcaggaac aagcgggcgctgctcgacgc actggccgaa gccatgctgg 540 cggagaatca tacgcattcg gtgccgagagccgacgacga ctggcgctca tttctgatcg 600 ggaatgcccg cagcttcagg caggcgctgctcgcctaccg cgatggcgcg cgcatccatg 660 ccggcacgcg accgggcgca ccgcagatggaaacggccga cgcgcagctt cgcttcctct 720 gcgaggcggg tttttcggcc ggggacgccgtcaatgcgct gatgacaatc agctacttca 780 ctgttggggc cgtgcttgag gagcaggccggcgacagcga tgccggcgag cgcggcggca 840 ccgttgaaca ggctccgctc tcgccgctgttgcgggccgc gatagacgcc ttcgacgaag 900 ccggtccgga cgcagcgttc gagcagggactcgcggtgat tgtcgatgga ttggcgaaaa 960 ggaggctcgt tgtcaggaac gttgaaggaccgagaaaggg tgacgattga tcaggaccgc 1020 tgccggagcg caacccactc actacagcagagccatgtag acaacatccc ctcccccttt 1080 ccaccgcgtc agacgcccgt agcagcccgctacgggcttt ttcatgccct gccctagcgt 1140 ccaagcctca cggccgcgct cggcctctctggcggccttc tggcgctctt ccgcttcctc 1200 gctcactgac tcgctgcgct cggtcgttcggctgcggcga gcggtatcag ctcactcaaa 1260 ggcggtaata cggttatcca cagaatcaggggataacgca ggaaagaaca tgtgagcaaa 1320 aggccagcaa aaggccagga accgtaaaaaggccgcgttg ctggcgtttt tccataggct 1380 ccgcccccct gacgagcatc acaaaaatcgacgctcaagt cagaggtggc gaaacccgac 1440 aggactataa agataccagg cgtttccccctggaagctcc ctcgtgcgct ctcctgttcc 1500 gaccctgccg cttaccggat acctgtccgcctttctccct tcgggaagcg tggcgctttt 1560 ccgctgcata accctgcttc ggggtcattatagcgatttt ttcggtatat ccatcctttt 1620 tcgcacgata tacaggattt tgccaaagggttcgtgtaga ctttccttgg tgtatccaac 1680 ggcgtcagcc gggcaggata ggtgaagtaggcccacccgc gagcgggtgt tccttcttca 1740 ctgtccctta ttcgcacctg gcggtgctcaacgggaatcc tgctctgcga ggctggccgg 1800 ctaccgccgg cgtaacagat gagggcaagcggatggctga tgaaaccaag ccaaccagga 1860 agggcagccc acctatcaag gtgtactgccttccagacga acgaagagcg attgaggaaa 1920 aggcggcggc ggccggcatg agcctgtcggcctacctgct ggccgtcggc cagggctaca 1980 aaatcacggg cgtcgtggac tatgagcacgtccgcgagct ggcccgcatc aatggcgacc 2040 tgggccgcct gggcggcctg ctgaaactctggctcaccga cgacccgcgc acggcgcggt 2100 tcggtgatgc cacgatcctc gccctgctggcgaagatcga agagaagcag gacgagcttg 2160 gcaaggtcat gatgggcgtg gtccgcccgagggcagagcc atgacttttt tagccgctaa 2220 aacggccggg gggtgcgcgt gattgccaagcacgtcccca tgcgctccat caagaagagc 2280 gacttcgcgg agctggtgaa gtacatcaccgacgagcaag gcaagaccga gcgcctttgc 2340 gacgctcacc gggctggttg ccctcgccgctgggctggcg gccgtctatg gccctgcaaa 2400 cgcgccagaa acgccgtcga agccgtgtgcgagacaccgc ggccgccggc gttgtggata 2460 cctcgcggaa aacttggccc tcactgacagatgaggggcg gacgttgaca cttgaggggc 2520 cgactcaccc ggcgcggcgt tgacagatgaggggcaggct cgatttcggc cggcgacgtg 2580 gagctggcca gcctcgcaaa tcggcgaaaacgcctgattt tacgcgagtt tcccacagat 2640 gatgtggaca agcctgggga taagtgccctgcggtattga cacttgaggg gcgcgactac 2700 tgacagatga ggggcgcgat ccttgacacttgaggggcag agtgctgaca gatgaggggc 2760 gcacctattg acatttgagg ggctgtccacaggcagaaaa tccagcattt gcaagggttt 2820 ccgcccgttt ttcggccacc gctaacctgtcttttaacct gcttttaaac caatatttat 2880 aaaccttgtt tttaaccagg gctgcgccctgtgcgcgtga ccgcgcacgc cgaagggggg 2940 tgccccccct tctcgaaccc tcccggcccgctaacgcggg cctcccatcc ccccaggggc 3000 tgcgcccctc ggccgcgaac ggcctcaccccaaaaatggc agcgctggca gtccttgcca 3060 ttgccgggat cggggcagta acgggatgggcgatcagccc gagcgcgacg cccggaagca 3120 ttgacgtgcc gcaggtgctg gcatcgacattcagcgacca ggtgccgggc agtgagggcg 3180 gcggcctggg tggcggcctg cccttcacttcggccgtcgg ggcattcacg gacttcatgg 3240 cggggccggc aatttttacc ttgggcattcttggcatagt ggtcgcgggt gccgtgctcg 3300 tgttcggggg tgcgataaac ccagcgaaccatttgaggtg ataggtaaga ttataccgag 3360 gtatgaaaac gagaattgga cctttacagaattactctat gaagcgccat atttaaaaag 3420 ctaccaagac gaagaggatg aagaggatgaggaggcagat tgccttgaat atattgacaa 3480 tactgataag ataatatatc ttttatatagaagatatcgc cgtatgtaag gatttcaggg 3540 ggcaaggcat aggcagcgcg cttatcaatatatctataga atgggcaaag cataaaaact 3600 tgcatggact aatgcttgaa acccaggacaataaccttat agcttgtaaa ttctatcata 3660 attgggtaat gactccaact tattgatagtgttttatgtt cagataatgc ccgatgactt 3720 tgtcatgcag ctccaccgat tttgagaacgacagcgactt ccgtcccagc cgtgccaggt 3780 gctgcctcag attcaggtta tgccgctcaattcgctgcgt atatcgcttg ctgattacgt 3840 gcagctttcc cttcaggcgg gattcatacagcggccagcc atccgtcatc catatcacca 3900 cgtcaaaggg tgacagcagg ctcataagacgccccagcgt cgccatagtg cgttcaccga 3960 atacgtgcgc aacaaccgtc ttccggagactgtcatacgc gtaaaacagc cagcgctggc 4020 gcgatttagc cccgacatag ccccactgttcgtccatttc cgcgcagacg atgacgtcac 4080 tgcccggctg tatgcgcgag gttaccgactgcggcctgag ttttttaagt gacgtaaaat 4140 cgtgttgagg ccaacgccca taatgcgggctgttgcccgg catccaacgc cattcatggc 4200 catatcaatg attttctggt gcgtaccgggttgagaagcg gtgtaagtga actgcagttg 4260 ccatgtttta cggcagtgag agcagagatagcgctgatgt ccggcggtgc ttttgccgtt 4320 acgcaccacc ccgtcagtag ctgaacaggagggacagctg atagacacag aagccactgg 4380 agcacctcaa aaacaccatc atacactaaatcagtaagtt ggcagcatca cccataattg 4440 tggtttcaaa atcggctccg tcgatactatgttatacgcc aactttgaaa acaactttga 4500 aaaagctgtt ttctggtatt taaggttttagaatgcaagg aacagtgaat tggagttcgt 4560 cttgttataa ttagcttctt ggggtatctttaaatactgt agaaaagagg aaggaaataa 4620 taaatggcta aaatgagaat atcaccggaattgaaaaaac tgatcgaaaa ataccgctgc 4680 gtaaaagata cggaaggaat gtctcctgctaaggtatata agctggtggg agaaaatgaa 4740 aacctatatt taaaaatgac ggacagccggtataaaggga ccacctatga tgtggaacgg 4800 gaaaaggaca tgatgctatg gctggaaggaaagctgcctg ttccaaaggt cctgcacttt 4860 gaacggcatg atggctggag caatctgctcatgagtgagg ccgatggcgt cctttgctcg 4920 gaagagtatg aagatgaaca aagccctgaaaagattatcg agctgtatgc ggagtgcatc 4980 aggctctttc actccatcga catatcggattgtccctata cgaatagctt agacagccgc 5040 ttagccgaat tggattactt actgaataacgatctggccg atgtggattg cgaaaactgg 5100 gaagaagaca ctccatttaa agatccgcgcgagctgtatg attttttaaa gacggaaaag 5160 cccgaagagg aacttgtctt ttcccacggcgacctgggag acagcaacat ctttgtgaaa 5220 gatggcaaag taagtggctt tattgatcttgggagaagcg gcagggcgga caagtggtat 5280 gacattgcct tctgcgtccg gtcgatcagggaggatatcg gggaagaaca gtatgtcgag 5340 ctattttttg acttactggg gatcaagcctgattgggaga aaataaaata ttatatttta 5400 ctggatgaat tgttttagta cctagatgtggcgcaacgat gccggcgaca agcaggagcg 5460 caccgacttc ttccgcatca agtgttttggctctcaggcc gaggcccacg gcaagtattt 5520 gggcaagggg tcgctggtat tcgtgcagggcaagattcgg aataccaagt acgagaagga 5580 cggccagacg gtctacggga ccgacttcattgccgataag gtggattatc tggacaccaa 5640 ggcaccaggc gggtcaaatc aggaataagggcacattgcc ccggcgtgag tcggggcaat 5700 cccgcaagga gggtgaatga atcggacgtttgaccggaag gcatacaggc aagaactgat 5760 cgacgcgggg ttttccgccg aggatgccgaaaccatcgca agccgcaccg tcatgcgtgc 5820 gccccgcgaa accttccagt ccgtcggctcgatggtccag caagctacgg ccaagatcga 5880 gcgcgacagc gtgcaactgg ctccccctgccctgcccgcg ccatcggccg ccgtggagcg 5940 ttcgcgtcgt ctcgaacagg aggcggcaggtttggcgaag tcgatgacca tcgacacgcg 6000 aggaactatg acgaccaaga agcgaaaaaccgccggcgag gacctggcaa aacaggtcag 6060 cgaggccaag caggccgcgt tgctgaaacacacgaagcag cagatcaagg aaatgcagct 6120 ttccttgttc gatattgcgc cgtggccggacacgatgcga gcgatgccaa acgacacggc 6180 ccgctctgcc ctgttcacca cgcgcaacaagaaaatcccg cgcgaggcgc tgcaaaacaa 6240 ggtcattttc cacgtcaaca aggacgtgaagatcacctac accggcgtcg agctgcgggc 6300 cgacgatgac gaactggtgt ggcagcaggtgttggagtac gcgaagcgca cccctatcgg 6360 cgagccgatc accttcacgt tctacgagctttgccaggac ctgggctggt cgatcaatgg 6420 ccggtattac acgaaggccg aggaatgcctgtcgcgccta caggcgacgg cgatgggctt 6480 cacgtccgac cgcgttgggc acctggaatcggtgtcgctg ctgcaccgct tccgcgtcct 6540 ggaccgtggc aagaaaacgt cccgttgccaggtcctgatc gacgaggaaa tcgtcgtgct 6600 gtttgctggc gaccactaca cgaaattcatatgggagaag taccgcaagc tgtcgccgac 6660 ggcccgacgg atgttcgact atttcagctcgcaccgggag ccgtacccgc tcaagctgga 6720 aaccttccgc ctcatgtgcg gatcggattccacccgcgtg aagaagtggc gcgagcaggt 6780 cggcgaagcc tgcgaagagt tgcgaggcagcggcctggtg gaacacgcct gggtcaatga 6840 tgacctggtg cattgcaaac gctagggccttgtggggtca gttccggctg ggggttcagc 6900 agccagcgct ttactggcat ttcaggaacaagcgggcact gctcgacgca cttgcttcgc 6960 tcagtatcgc tcgggacgca cggcgcgctctacgaactgc cgataaacag aggattaaaa 7020 ttgacaattg tgattaaggc tcagattcgacggcttggag cggccgacgt gcaggatttc 7080 cgcgagatcc gattgtcggc cctgaagaaagctccagaga tgttcgggtc cgtttacgag 7140 cacgaggaga aaaagcccat ggaggcgttcgctgaacggt tgcgagatgc cgtggcattc 7200 ggcgcctaca tcgacggcga gatcattgggctgtcggtct tcaaacagga ggacggcccc 7260 aaggacgctc acaaggcgca tctgtccggcgttttcgtgg agcccgaaca gcgaggccga 7320 ggggtcgccg gtatgctgct gcgggcgttgccggcgggtt tattgctcgt gatgatcgtc 7380 cgacagattc caacgggaat ctggtggatgcgcatcttca tcctcggcgc acttaatatt 7440 tcgctattct ggagcttgtt gtttatttcggtctaccgcc tgccgggcgg ggtcgcggcg 7500 acggtaggcg ctgtgcagcc gctgatggtcgtgttcatct ctgccgctct gctaggtagc 7560 ccgatacgat tgatggcggt cctgggggctatttgcggaa ctgcgggcgt ggcgctgttg 7620 gtgttgacac caaacgcagc gctagatcctgtcggcgtcg cagcgggcct ggcgggggcg 7680 gtttccatgg cgttcggaac cgtgctgacccgcaagtggc aacctcccgt gcctctgctc 7740 acctttaccg cctggcaact ggcggccggaggacttctgc tcgttccagt agctttagtg 7800 tttgatccgc caatcccgat gcctacaggaaccaatgttc tcggcctggc gtggctcggc 7860 ctgatcggag cgggtttaac ctacttcctttggttccggg ggatctcgcg actcgaacct 7920 acagttgttt ccttactggg ctttctcagccccagatctg gggtcgatca gccggggatg 7980 catcaggccg acagtcggaa cttcgggtccccgacctgta ccattcggtg agcaatggat 8040 aggggagttg atatcgtcaa cgttcacttctaaagaaata gcgccactca gcttcctcag 8100 cggctttatc cagcgatttc ctattatgtcggcatagttc tcaagatcga cagcctgtca 8160 cggttaagcg agaaatgaat aagaaggctgataattcgga tctctgcgag ggagatgata 8220 tttgatcaca ggcagcaacg ctctgtcatcgttacaatca acatgctacc ctccgcgaga 8280 tcatccgtgt ttcaaacccg gcagcttagttgccgttctt ccgaatagca tcggtaacat 8340 gagcaaagtc tgccgcctta caacggctctcccgctgacg ccgtcccgga ctgatgggct 8400 gcctgtatcg agtggtgatt ttgtgccgagctgccggtcg gggagctgtt ggctggctgg 8460 tggcaggata tattgtggtg taaacaaattgacgcttaga caacttaata acacattgcg 8520 gacgttttta atgtactggg gtggtttttcttttcaccag tgagacgggc aacagctgat 8580 tgcccttcac cgcctggccc tgagagagttgcagcaagcg gtccacgctg gtttgcccca 8640 gcaggcgaaa atcctgtttg atggtggttccgaaatcggc aaaatccctt ataaatcaaa 8700 agaatagccc gagatagggt tgagtgttgttccagtttgg aacaagagtc cactattaaa 8760 gaacgtggac tccaacgtca aagggcgaaaaaccgtctat cagggcgatg gcccactacg 8820 tgaaccatca cccaaatcaa gttttttggggtcgaggtgc cgtaaagcac taaatcggaa 8880 ccctaaaggg agcccccgat ttagagcttgacggggaaag ccggcgaacg tggcgagaaa 8940 ggaagggaag aaagcgaaag gagcgggcgccattcaggct gcgcaactgt tgggaagggc 9000 gatcggtgcg ggcctcttcg ctattacgccagctggcgaa agggggatgt gctgcaaggc 9060 gattaagttg ggtaacgcca gggttttcccagtcacgacg ttgtaaaacg acggccagtg 9120 aattaattcc catcttgaaa gaaatatagtttaaatattt attgataaaa taacaagtca 9180 ggtattatag tccaagcaaa aacataaatttattgatgca agtttaaatt cagaaatatt 9240 tcaataactg attatatcag ctggtacattgccgtagatg aaagactgag tgcgatatta 9300 tgtgtaatac ataaattgat gatatagctagcttagctca tcgggggatc cgtcgaagct 9360 agcttgggtc ccgctcagaa gaactcgtcaagaaggcgat agaaggcgat gcgctgcgaa 9420 tcgggagcgg cgataccgta aagcacgaggaagcggtcag cccattcgcc gccaagctct 9480 tcagcaatat cacgggtagc caacgctatgtcctgatagc ggtccgccac acccagccgg 9540 ccacagtcga tgaatccaga aaagcggccattttccacca tgatattcgg caagcaggca 9600 tcgccatggg tcacgacgag atcctcgccgtcgggcatgc gcgccttgag cctggcgaac 9660 agttcggctg gcgcgagccc ctgatgctcttcgtccagat catcctgatc gacaagaccg 9720 gcttccatcc gagtacgtgc tcgctcgatgcgatgtttcg cttggtggtc gaatgggcag 9780 gtagccggat caagcgtatg cagccgccgcattgcatcag ccatgatgga tactttctcg 9840 gcaggagcaa ggtgagatga caggagatcctgccccggca cttcgcccaa tagcagccag 9900 tcccttcccg cttcagtgac aacgtcgagcacagctgcgc aaggaacgcc cgtcgtggcc 9960 agccacgata gccgcgctgc ctcgtcctgcagttcattca gggcaccgga caggtcggtc 10020 ttgacaaaaa gaaccgggcg cccctgcgctgacagccgga acacggcggc atcagagcag 10080 ccgattgtct gttgtgccca gtcatagccgaatagcctct ccacccaagc ggccggagaa 10140 cctgcgtgca atccatcttg ttcaatccaagctcccatgg gccctcgact agagtcgaga 10200 tctggattga gagtgaatat gagactctaattggataccg aggggaattt atggaacgtc 10260 agtggagcat ttttgacaag aaatatttgctagctgatag tgaccttagg cgacttttga 10320 acgcgcaata atggtttctg acgtatgtgcttagctcatt aaactccaga aacccgcggc 10380 tgagtggctc cttcaacgtt gcggttctgtcagttccaaa cgtaaaacgg cttgtcccgc 10440 gtcatcggcg ggggtcataa cgtgactcccttaattctcc gctcatgatc ttgatcccct 10500 gcgccatcag atccttggcg gcaagaaagccatccagttt actttgcagg gcttcccaac 10560 cttaccagag ggcgccccag ctggcaattccggttcgctt gctgtccata aaaccgccca 10620 gtctagctat cgccatgtaa gcccactgcaagctacctgc tttctctttg cgcttgcgtt 10680 ttcccttgtc cagatagccc agtagctgacattcatccgg ggtcagcacc gtttctgcgg 10740 actggctttc tacgtgttcc gcttcctttagcagcccttg cgccctgagt gcttgcggca 10800 gcgtgaagct tgcatgcctg caggtcgacggcgcgccgag ctcctcgagc aaatttacac 10860 attgccacta aacgtctaaa cccttgtaatttgtttttgt tttactatgt gtgttatgta 10920 tttgatttgc gataaatttt tatatttggtactaaattta taacaccttt tatgctaacg 10980 tttgccaaca cttagcaatt tgcaagttgattaattgatt ctaaattatt tttgtcttct 11040 aaatacatat actaatcaac tggaaatgtaaatatttgct aatatttcta ctataggaga 11100 attaaagtga gtgaatatgg taccacaaggtttggagatt taattgttgc aatgctgcat 11160 ggatggcata tacaccaaac attcaataattcttgaggat aataatggta ccacacaaga 11220 tttgaggtgc atgaacgtca cgtggacaaaaggtttagta atttttcaag acaacaatgt 11280 taccacacac aagttttgag gtgcatgcatggatgccctg tggaaagttt aaaaatattt 11340 tggaaatgat ttgcatggaa gccatgtgtaaaaccatgac atccacttgg aggatgcaat 11400 aatgaagaaa actacaaatt tacatgcaactagttatgca tgtagtctat ataatgagga 11460 ttttgcaata ctttcattca tacacactcactaagtttta cacgattata atttcttcat 11520 agccagccca ccgcggtggg cggccgcctgcagtctagaa ggcctcctgc tttaatgaga 11580 tatgcgagac gcctatgatc gcatgatatttgctttcaat tctgttgtgc acgttgtaaa 11640 aaacctgagc atgtgtagct cagatccttaccgccggttt cggttcattc taatgaatat 11700 atcacccgtt actatcgtat ttttatgaataatattctcc gttcaattta ctgattgtcc 11760 gtcgagcaaa tttacacatt gccactaaacgtctaaaccc ttgtaatttg tttttgtttt 11820 actatgtgtg ttatgtattt gatttgcgataaatttttat atttggtact aaatttataa 11880 caccttttat gctaacgttt gccaacacttagcaatttgc aagttgatta attgattcta 11940 aattattttt gtcttctaaa tacatatactaatcaactgg aaatgtaaat atttgctaat 12000 atttctacta taggagaatt aaagtgagtgaatatggtac cacaaggttt ggagatttaa 12060 ttgttgcaat gctgcatgga tggcatatacaccaaacatt caataattct tgaggataat 12120 aatggtacca cacaagattt gaggtgcatgaacgtcacgt ggacaaaagg tttagtaatt 12180 tttcaagaca acaatgttac cacacacaagttttgaggtg catgcatgga tgccctgtgg 12240 aaagtttaaa aatattttgg aaatgatttgcatggaagcc atgtgtaaaa ccatgacatc 12300 cacttggagg atgcaataat gaagaaaactacaaatttac atgcaactag ttatgcatgt 12360 agtctatata atgaggattt tgcaatactttcattcatac acactcacta agttttacac 12420 gattataatt tcttcatagc cagcggatccgatatcgggc ccgctagcgt taaccctgct 12480 ttaatgagat atgcgagacg cctatgatcgcatgatattt gctttcaatt ctgttgtgca 12540 cgttgtaaaa aacctgagca tgtgtagctcagatccttac cgccggtttc ggttcattct 12600 aatgaatata tcacccgtta ctatcgtatttttatgaata atattctccg ttcaatttac 12660 tgattgtccg tcgagcaaat ttacacattgccactaaacg tctaaaccct tgtaatttgt 12720 ttttgtttta ctatgtgtgt tatgtatttgatttgcgata aatttttata tttggtacta 12780 aatttataac accttttatg ctaacgtttgccaacactta gcaatttgca agttgattaa 12840 ttgattctaa attatttttg tcttctaaatacatatacta atcaactgga aatgtaaata 12900 tttgctaata tttctactat aggagaattaaagtgagtga atatggtacc acaaggtttg 12960 gagatttaat tgttgcaatg ctgcatggatggcatataca ccaaacattc aataattctt 13020 gaggataata atggtaccac acaagatttgaggtgcatga acgtcacgtg gacaaaaggt 13080 ttagtaattt ttcaagacaa caatgttaccacacacaagt tttgaggtgc atgcatggat 13140 gccctgtgga aagtttaaaa atattttggaaatgatttgc atggaagcca tgtgtaaaac 13200 catgacatcc acttggagga tgcaataatgaagaaaacta caaatttaca tgcaactagt 13260 tatgcatgta gtctatataa tgaggattttgcaatacttt cattcataca cactcactaa 13320 gttttacacg attataattt cttcatagccagcagatctg ccggcatcga tcccgggcca 13380 tggcctgctt taatgagata tgcgagacgcctatgatcgc atgatatttg ctttcaattc 13440 tgttgtgcac gttgtaaaaa acctgagcatgtgtagctca gatccttacc gccggtttcg 13500 gttcattcta atgaatatat cacccgttactatcgtattt ttatgaataa tattctccgt 13560 tcaatttact gattgtccgt cgacgagctcggcgcgcctc tagaggatcg atgaattcag 13620 atcggctgag tggctccttc aacgttgcggttctgtcagt tccaaacgta aaacggcttg 13680 tcccgcgtca tcggcggggg tcataacgtgactcccttaa ttctccgctc atgatcagat 13740 tgtcgtttcc cgccttcagt ttaaactatcagtgtttgac aggatatatt ggcgggtaaa 13800 cctaagagaa aagagcgttt attagaataatcggatattt aaaagggcgt gaaaaggttt 13860 atccttcgtc catttgtatg tgcatgccaaccacagggtt cccca 13905 25 15430 DNA Unknown Plant expression vector withtwo promoter- terminator expression cassettes; Physcomitrella patenselongase and desaturase are inserted 25 gatctggcgc cggccagcga gacgagcaagattggccgcc gcccgaaacg atccgacagc 60 gcgcccagca caggtgcgca ggcaaattgcaccaacgcat acagcgccag cagaatgcca 120 tagtgggcgg tgacgtcgtt cgagtgaaccagatcgcgca ggaggcccgg cagcaccggc 180 ataatcaggc cgatgccgac agcgtcgagcgcgacagtgc tcagaattac gatcaggggt 240 atgttgggtt tcacgtctgg cctccggaccagcctccgct ggtccgattg aacgcgcgga 300 ttctttatca ctgataagtt ggtggacatattatgtttat cagtgataaa gtgtcaagca 360 tgacaaagtt gcagccgaat acagtgatccgtgccgccct ggacctgttg aacgaggtcg 420 gcgtagacgg tctgacgaca cgcaaactggcggaacggtt gggggttcag cagccggcgc 480 tttactggca cttcaggaac aagcgggcgctgctcgacgc actggccgaa gccatgctgg 540 cggagaatca tacgcattcg gtgccgagagccgacgacga ctggcgctca tttctgatcg 600 ggaatgcccg cagcttcagg caggcgctgctcgcctaccg cgatggcgcg cgcatccatg 660 ccggcacgcg accgggcgca ccgcagatggaaacggccga cgcgcagctt cgcttcctct 720 gcgaggcggg tttttcggcc ggggacgccgtcaatgcgct gatgacaatc agctacttca 780 ctgttggggc cgtgcttgag gagcaggccggcgacagcga tgccggcgag cgcggcggca 840 ccgttgaaca ggctccgctc tcgccgctgttgcgggccgc gatagacgcc ttcgacgaag 900 ccggtccgga cgcagcgttc gagcagggactcgcggtgat tgtcgatgga ttggcgaaaa 960 ggaggctcgt tgtcaggaac gttgaaggaccgagaaaggg tgacgattga tcaggaccgc 1020 tgccggagcg caacccactc actacagcagagccatgtag acaacatccc ctcccccttt 1080 ccaccgcgtc agacgcccgt agcagcccgctacgggcttt ttcatgccct gccctagcgt 1140 ccaagcctca cggccgcgct cggcctctctggcggccttc tggcgctctt ccgcttcctc 1200 gctcactgac tcgctgcgct cggtcgttcggctgcggcga gcggtatcag ctcactcaaa 1260 ggcggtaata cggttatcca cagaatcaggggataacgca ggaaagaaca tgtgagcaaa 1320 aggccagcaa aaggccagga accgtaaaaaggccgcgttg ctggcgtttt tccataggct 1380 ccgcccccct gacgagcatc acaaaaatcgacgctcaagt cagaggtggc gaaacccgac 1440 aggactataa agataccagg cgtttccccctggaagctcc ctcgtgcgct ctcctgttcc 1500 gaccctgccg cttaccggat acctgtccgcctttctccct tcgggaagcg tggcgctttt 1560 ccgctgcata accctgcttc ggggtcattatagcgatttt ttcggtatat ccatcctttt 1620 tcgcacgata tacaggattt tgccaaagggttcgtgtaga ctttccttgg tgtatccaac 1680 ggcgtcagcc gggcaggata ggtgaagtaggcccacccgc gagcgggtgt tccttcttca 1740 ctgtccctta ttcgcacctg gcggtgctcaacgggaatcc tgctctgcga ggctggccgg 1800 ctaccgccgg cgtaacagat gagggcaagcggatggctga tgaaaccaag ccaaccagga 1860 agggcagccc acctatcaag gtgtactgccttccagacga acgaagagcg attgaggaaa 1920 aggcggcggc ggccggcatg agcctgtcggcctacctgct ggccgtcggc cagggctaca 1980 aaatcacggg cgtcgtggac tatgagcacgtccgcgagct ggcccgcatc aatggcgacc 2040 tgggccgcct gggcggcctg ctgaaactctggctcaccga cgacccgcgc acggcgcggt 2100 tcggtgatgc cacgatcctc gccctgctggcgaagatcga agagaagcag gacgagcttg 2160 gcaaggtcat gatgggcgtg gtccgcccgagggcagagcc atgacttttt tagccgctaa 2220 aacggccggg gggtgcgcgt gattgccaagcacgtcccca tgcgctccat caagaagagc 2280 gacttcgcgg agctggtgaa gtacatcaccgacgagcaag gcaagaccga gcgcctttgc 2340 gacgctcacc gggctggttg ccctcgccgctgggctggcg gccgtctatg gccctgcaaa 2400 cgcgccagaa acgccgtcga agccgtgtgcgagacaccgc ggccgccggc gttgtggata 2460 cctcgcggaa aacttggccc tcactgacagatgaggggcg gacgttgaca cttgaggggc 2520 cgactcaccc ggcgcggcgt tgacagatgaggggcaggct cgatttcggc cggcgacgtg 2580 gagctggcca gcctcgcaaa tcggcgaaaacgcctgattt tacgcgagtt tcccacagat 2640 gatgtggaca agcctgggga taagtgccctgcggtattga cacttgaggg gcgcgactac 2700 tgacagatga ggggcgcgat ccttgacacttgaggggcag agtgctgaca gatgaggggc 2760 gcacctattg acatttgagg ggctgtccacaggcagaaaa tccagcattt gcaagggttt 2820 ccgcccgttt ttcggccacc gctaacctgtcttttaacct gcttttaaac caatatttat 2880 aaaccttgtt tttaaccagg gctgcgccctgtgcgcgtga ccgcgcacgc cgaagggggg 2940 tgccccccct tctcgaaccc tcccggcccgctaacgcggg cctcccatcc ccccaggggc 3000 tgcgcccctc ggccgcgaac ggcctcaccccaaaaatggc agcgctggca gtccttgcca 3060 ttgccgggat cggggcagta acgggatgggcgatcagccc gagcgcgacg cccggaagca 3120 ttgacgtgcc gcaggtgctg gcatcgacattcagcgacca ggtgccgggc agtgagggcg 3180 gcggcctggg tggcggcctg cccttcacttcggccgtcgg ggcattcacg gacttcatgg 3240 cggggccggc aatttttacc ttgggcattcttggcatagt ggtcgcgggt gccgtgctcg 3300 tgttcggggg tgcgataaac ccagcgaaccatttgaggtg ataggtaaga ttataccgag 3360 gtatgaaaac gagaattgga cctttacagaattactctat gaagcgccat atttaaaaag 3420 ctaccaagac gaagaggatg aagaggatgaggaggcagat tgccttgaat atattgacaa 3480 tactgataag ataatatatc ttttatatagaagatatcgc cgtatgtaag gatttcaggg 3540 ggcaaggcat aggcagcgcg cttatcaatatatctataga atgggcaaag cataaaaact 3600 tgcatggact aatgcttgaa acccaggacaataaccttat agcttgtaaa ttctatcata 3660 attgggtaat gactccaact tattgatagtgttttatgtt cagataatgc ccgatgactt 3720 tgtcatgcag ctccaccgat tttgagaacgacagcgactt ccgtcccagc cgtgccaggt 3780 gctgcctcag attcaggtta tgccgctcaattcgctgcgt atatcgcttg ctgattacgt 3840 gcagctttcc cttcaggcgg gattcatacagcggccagcc atccgtcatc catatcacca 3900 cgtcaaaggg tgacagcagg ctcataagacgccccagcgt cgccatagtg cgttcaccga 3960 atacgtgcgc aacaaccgtc ttccggagactgtcatacgc gtaaaacagc cagcgctggc 4020 gcgatttagc cccgacatag ccccactgttcgtccatttc cgcgcagacg atgacgtcac 4080 tgcccggctg tatgcgcgag gttaccgactgcggcctgag ttttttaagt gacgtaaaat 4140 cgtgttgagg ccaacgccca taatgcgggctgttgcccgg catccaacgc cattcatggc 4200 catatcaatg attttctggt gcgtaccgggttgagaagcg gtgtaagtga actgcagttg 4260 ccatgtttta cggcagtgag agcagagatagcgctgatgt ccggcggtgc ttttgccgtt 4320 acgcaccacc ccgtcagtag ctgaacaggagggacagctg atagacacag aagccactgg 4380 agcacctcaa aaacaccatc atacactaaatcagtaagtt ggcagcatca cccataattg 4440 tggtttcaaa atcggctccg tcgatactatgttatacgcc aactttgaaa acaactttga 4500 aaaagctgtt ttctggtatt taaggttttagaatgcaagg aacagtgaat tggagttcgt 4560 cttgttataa ttagcttctt ggggtatctttaaatactgt agaaaagagg aaggaaataa 4620 taaatggcta aaatgagaat atcaccggaattgaaaaaac tgatcgaaaa ataccgctgc 4680 gtaaaagata cggaaggaat gtctcctgctaaggtatata agctggtggg agaaaatgaa 4740 aacctatatt taaaaatgac ggacagccggtataaaggga ccacctatga tgtggaacgg 4800 gaaaaggaca tgatgctatg gctggaaggaaagctgcctg ttccaaaggt cctgcacttt 4860 gaacggcatg atggctggag caatctgctcatgagtgagg ccgatggcgt cctttgctcg 4920 gaagagtatg aagatgaaca aagccctgaaaagattatcg agctgtatgc ggagtgcatc 4980 aggctctttc actccatcga catatcggattgtccctata cgaatagctt agacagccgc 5040 ttagccgaat tggattactt actgaataacgatctggccg atgtggattg cgaaaactgg 5100 gaagaagaca ctccatttaa agatccgcgcgagctgtatg attttttaaa gacggaaaag 5160 cccgaagagg aacttgtctt ttcccacggcgacctgggag acagcaacat ctttgtgaaa 5220 gatggcaaag taagtggctt tattgatcttgggagaagcg gcagggcgga caagtggtat 5280 gacattgcct tctgcgtccg gtcgatcagggaggatatcg gggaagaaca gtatgtcgag 5340 ctattttttg acttactggg gatcaagcctgattgggaga aaataaaata ttatatttta 5400 ctggatgaat tgttttagta cctagatgtggcgcaacgat gccggcgaca agcaggagcg 5460 caccgacttc ttccgcatca agtgttttggctctcaggcc gaggcccacg gcaagtattt 5520 gggcaagggg tcgctggtat tcgtgcagggcaagattcgg aataccaagt acgagaagga 5580 cggccagacg gtctacggga ccgacttcattgccgataag gtggattatc tggacaccaa 5640 ggcaccaggc gggtcaaatc aggaataagggcacattgcc ccggcgtgag tcggggcaat 5700 cccgcaagga gggtgaatga atcggacgtttgaccggaag gcatacaggc aagaactgat 5760 cgacgcgggg ttttccgccg aggatgccgaaaccatcgca agccgcaccg tcatgcgtgc 5820 gccccgcgaa accttccagt ccgtcggctcgatggtccag caagctacgg ccaagatcga 5880 gcgcgacagc gtgcaactgg ctccccctgccctgcccgcg ccatcggccg ccgtggagcg 5940 ttcgcgtcgt ctcgaacagg aggcggcaggtttggcgaag tcgatgacca tcgacacgcg 6000 aggaactatg acgaccaaga agcgaaaaaccgccggcgag gacctggcaa aacaggtcag 6060 cgaggccaag caggccgcgt tgctgaaacacacgaagcag cagatcaagg aaatgcagct 6120 ttccttgttc gatattgcgc cgtggccggacacgatgcga gcgatgccaa acgacacggc 6180 ccgctctgcc ctgttcacca cgcgcaacaagaaaatcccg cgcgaggcgc tgcaaaacaa 6240 ggtcattttc cacgtcaaca aggacgtgaagatcacctac accggcgtcg agctgcgggc 6300 cgacgatgac gaactggtgt ggcagcaggtgttggagtac gcgaagcgca cccctatcgg 6360 cgagccgatc accttcacgt tctacgagctttgccaggac ctgggctggt cgatcaatgg 6420 ccggtattac acgaaggccg aggaatgcctgtcgcgccta caggcgacgg cgatgggctt 6480 cacgtccgac cgcgttgggc acctggaatcggtgtcgctg ctgcaccgct tccgcgtcct 6540 ggaccgtggc aagaaaacgt cccgttgccaggtcctgatc gacgaggaaa tcgtcgtgct 6600 gtttgctggc gaccactaca cgaaattcatatgggagaag taccgcaagc tgtcgccgac 6660 ggcccgacgg atgttcgact atttcagctcgcaccgggag ccgtacccgc tcaagctgga 6720 aaccttccgc ctcatgtgcg gatcggattccacccgcgtg aagaagtggc gcgagcaggt 6780 cggcgaagcc tgcgaagagt tgcgaggcagcggcctggtg gaacacgcct gggtcaatga 6840 tgacctggtg cattgcaaac gctagggccttgtggggtca gttccggctg ggggttcagc 6900 agccagcgct ttactggcat ttcaggaacaagcgggcact gctcgacgca cttgcttcgc 6960 tcagtatcgc tcgggacgca cggcgcgctctacgaactgc cgataaacag aggattaaaa 7020 ttgacaattg tgattaaggc tcagattcgacggcttggag cggccgacgt gcaggatttc 7080 cgcgagatcc gattgtcggc cctgaagaaagctccagaga tgttcgggtc cgtttacgag 7140 cacgaggaga aaaagcccat ggaggcgttcgctgaacggt tgcgagatgc cgtggcattc 7200 ggcgcctaca tcgacggcga gatcattgggctgtcggtct tcaaacagga ggacggcccc 7260 aaggacgctc acaaggcgca tctgtccggcgttttcgtgg agcccgaaca gcgaggccga 7320 ggggtcgccg gtatgctgct gcgggcgttgccggcgggtt tattgctcgt gatgatcgtc 7380 cgacagattc caacgggaat ctggtggatgcgcatcttca tcctcggcgc acttaatatt 7440 tcgctattct ggagcttgtt gtttatttcggtctaccgcc tgccgggcgg ggtcgcggcg 7500 acggtaggcg ctgtgcagcc gctgatggtcgtgttcatct ctgccgctct gctaggtagc 7560 ccgatacgat tgatggcggt cctgggggctatttgcggaa ctgcgggcgt ggcgctgttg 7620 gtgttgacac caaacgcagc gctagatcctgtcggcgtcg cagcgggcct ggcgggggcg 7680 gtttccatgg cgttcggaac cgtgctgacccgcaagtggc aacctcccgt gcctctgctc 7740 acctttaccg cctggcaact ggcggccggaggacttctgc tcgttccagt agctttagtg 7800 tttgatccgc caatcccgat gcctacaggaaccaatgttc tcggcctggc gtggctcggc 7860 ctgatcggag cgggtttaac ctacttcctttggttccggg ggatctcgcg actcgaacct 7920 acagttgttt ccttactggg ctttctcagccccagatctg gggtcgatca gccggggatg 7980 catcaggccg acagtcggaa cttcgggtccccgacctgta ccattcggtg agcaatggat 8040 aggggagttg atatcgtcaa cgttcacttctaaagaaata gcgccactca gcttcctcag 8100 cggctttatc cagcgatttc ctattatgtcggcatagttc tcaagatcga cagcctgtca 8160 cggttaagcg agaaatgaat aagaaggctgataattcgga tctctgcgag ggagatgata 8220 tttgatcaca ggcagcaacg ctctgtcatcgttacaatca acatgctacc ctccgcgaga 8280 tcatccgtgt ttcaaacccg gcagcttagttgccgttctt ccgaatagca tcggtaacat 8340 gagcaaagtc tgccgcctta caacggctctcccgctgacg ccgtcccgga ctgatgggct 8400 gcctgtatcg agtggtgatt ttgtgccgagctgccggtcg gggagctgtt ggctggctgg 8460 tggcaggata tattgtggtg taaacaaattgacgcttaga caacttaata acacattgcg 8520 gacgttttta atgtactggg gtggtttttcttttcaccag tgagacgggc aacagctgat 8580 tgcccttcac cgcctggccc tgagagagttgcagcaagcg gtccacgctg gtttgcccca 8640 gcaggcgaaa atcctgtttg atggtggttccgaaatcggc aaaatccctt ataaatcaaa 8700 agaatagccc gagatagggt tgagtgttgttccagtttgg aacaagagtc cactattaaa 8760 gaacgtggac tccaacgtca aagggcgaaaaaccgtctat cagggcgatg gcccactacg 8820 tgaaccatca cccaaatcaa gttttttggggtcgaggtgc cgtaaagcac taaatcggaa 8880 ccctaaaggg agcccccgat ttagagcttgacggggaaag ccggcgaacg tggcgagaaa 8940 ggaagggaag aaagcgaaag gagcgggcgccattcaggct gcgcaactgt tgggaagggc 9000 gatcggtgcg ggcctcttcg ctattacgccagctggcgaa agggggatgt gctgcaaggc 9060 gattaagttg ggtaacgcca gggttttcccagtcacgacg ttgtaaaacg acggccagtg 9120 aattaattcc catcttgaaa gaaatatagtttaaatattt attgataaaa taacaagtca 9180 ggtattatag tccaagcaaa aacataaatttattgatgca agtttaaatt cagaaatatt 9240 tcaataactg attatatcag ctggtacattgccgtagatg aaagactgag tgcgatatta 9300 tgtgtaatac ataaattgat gatatagctagcttagctca tcgggggatc cgtcgaagct 9360 agcttgggtc ccgctcagaa gaactcgtcaagaaggcgat agaaggcgat gcgctgcgaa 9420 tcgggagcgg cgataccgta aagcacgaggaagcggtcag cccattcgcc gccaagctct 9480 tcagcaatat cacgggtagc caacgctatgtcctgatagc ggtccgccac acccagccgg 9540 ccacagtcga tgaatccaga aaagcggccattttccacca tgatattcgg caagcaggca 9600 tcgccatggg tcacgacgag atcctcgccgtcgggcatgc gcgccttgag cctggcgaac 9660 agttcggctg gcgcgagccc ctgatgctcttcgtccagat catcctgatc gacaagaccg 9720 gcttccatcc gagtacgtgc tcgctcgatgcgatgtttcg cttggtggtc gaatgggcag 9780 gtagccggat caagcgtatg cagccgccgcattgcatcag ccatgatgga tactttctcg 9840 gcaggagcaa ggtgagatga caggagatcctgccccggca cttcgcccaa tagcagccag 9900 tcccttcccg cttcagtgac aacgtcgagcacagctgcgc aaggaacgcc cgtcgtggcc 9960 agccacgata gccgcgctgc ctcgtcctgcagttcattca gggcaccgga caggtcggtc 10020 ttgacaaaaa gaaccgggcg cccctgcgctgacagccgga acacggcggc atcagagcag 10080 ccgattgtct gttgtgccca gtcatagccgaatagcctct ccacccaagc ggccggagaa 10140 cctgcgtgca atccatcttg ttcaatccaagctcccatgg gccctcgact agagtcgaga 10200 tctggattga gagtgaatat gagactctaattggataccg aggggaattt atggaacgtc 10260 agtggagcat ttttgacaag aaatatttgctagctgatag tgaccttagg cgacttttga 10320 acgcgcaata atggtttctg acgtatgtgcttagctcatt aaactccaga aacccgcggc 10380 tgagtggctc cttcaacgtt gcggttctgtcagttccaaa cgtaaaacgg cttgtcccgc 10440 gtcatcggcg ggggtcataa cgtgactcccttaattctcc gctcatgatc ttgatcccct 10500 gcgccatcag atccttggcg gcaagaaagccatccagttt actttgcagg gcttcccaac 10560 cttaccagag ggcgccccag ctggcaattccggttcgctt gctgtccata aaaccgccca 10620 gtctagctat cgccatgtaa gcccactgcaagctacctgc tttctctttg cgcttgcgtt 10680 ttcccttgtc cagatagccc agtagctgacattcatccgg ggtcagcacc gtttctgcgg 10740 actggctttc tacgtgttcc gcttcctttagcagcccttg cgccctgagt gcttgcggca 10800 gcgtgaagct tgcatgcctg caggtcgacggcgcgccgag ctcctcgagc aaatttacac 10860 attgccacta aacgtctaaa cccttgtaatttgtttttgt tttactatgt gtgttatgta 10920 tttgatttgc gataaatttt tatatttggtactaaattta taacaccttt tatgctaacg 10980 tttgccaaca cttagcaatt tgcaagttgattaattgatt ctaaattatt tttgtcttct 11040 aaatacatat actaatcaac tggaaatgtaaatatttgct aatatttcta ctataggaga 11100 attaaagtga gtgaatatgg taccacaaggtttggagatt taattgttgc aatgctgcat 11160 ggatggcata tacaccaaac attcaataattcttgaggat aataatggta ccacacaaga 11220 tttgaggtgc atgaacgtca cgtggacaaaaggtttagta atttttcaag acaacaatgt 11280 taccacacac aagttttgag gtgcatgcatggatgccctg tggaaagttt aaaaatattt 11340 tggaaatgat ttgcatggaa gccatgtgtaaaaccatgac atccacttgg aggatgcaat 11400 aatgaagaaa actacaaatt tacatgcaactagttatgca tgtagtctat ataatgagga 11460 ttttgcaata ctttcattca tacacactcactaagtttta cacgattata atttcttcat 11520 agccagccca ccgcggtgga aa atg gaggtc gtg gag aga ttc tac ggt gag 11572 Met Glu Val Val Glu Arg Phe TyrGly Glu 1 5 10 ttg gat ggg aag gtc tcg cag ggc gtg aat gca ttg ctg ggtagt ttt 11620 Leu Asp Gly Lys Val Ser Gln Gly Val Asn Ala Leu Leu GlySer Phe 15 20 25 ggg gtg gag ttg acg gat acg ccc act acc aaa ggc ttg cccctc gtt 11668 Gly Val Glu Leu Thr Asp Thr Pro Thr Thr Lys Gly Leu ProLeu Val 30 35 40 gac agt ccc aca ccc atc gtc ctc ggt gtt tct gta tac ttgact att 11716 Asp Ser Pro Thr Pro Ile Val Leu Gly Val Ser Val Tyr LeuThr Ile 45 50 55 gtc att gga ggg ctt ttg tgg ata aag gcc agg gat ctg aaaccg cgc 11764 Val Ile Gly Gly Leu Leu Trp Ile Lys Ala Arg Asp Leu LysPro Arg 60 65 70 gcc tcg gag cca ttt ttg ctc caa gct ttg gtg ctt gtg cacaac ctg 11812 Ala Ser Glu Pro Phe Leu Leu Gln Ala Leu Val Leu Val HisAsn Leu 75 80 85 90 ttc tgt ttt gcg ctc agt ctg tat atg tgc gtg ggc atcgct tat cag 11860 Phe Cys Phe Ala Leu Ser Leu Tyr Met Cys Val Gly IleAla Tyr Gln 95 100 105 gct att acc tgg cgg tac tct ctc tgg ggc aat gcatac aat cct aaa 11908 Ala Ile Thr Trp Arg Tyr Ser Leu Trp Gly Asn AlaTyr Asn Pro Lys 110 115 120 cat aaa gag atg gcg att ctg gta tac ttg ttctac atg tct aag tac 11956 His Lys Glu Met Ala Ile Leu Val Tyr Leu PheTyr Met Ser Lys Tyr 125 130 135 gtg gaa ttc atg gat acc gtt atc atg atactg aag cgc agc acc agg 12004 Val Glu Phe Met Asp Thr Val Ile Met IleLeu Lys Arg Ser Thr Arg 140 145 150 caa ata agc ttc ctc cac gtt tat catcat tct tca att tcc ctc att 12052 Gln Ile Ser Phe Leu His Val Tyr HisHis Ser Ser Ile Ser Leu Ile 155 160 165 170 tgg tgg gct att gct cat cacgct cct ggc ggt gaa gca tat tgg tct 12100 Trp Trp Ala Ile Ala His HisAla Pro Gly Gly Glu Ala Tyr Trp Ser 175 180 185 gcg gct ctg aac tca ggagtg cat gtt ctc atg tat gcg tat tac ttc 12148 Ala Ala Leu Asn Ser GlyVal His Val Leu Met Tyr Ala Tyr Tyr Phe 190 195 200 ttg gct gcc tgc cttcga agt agc cca aag tta aaa aat aag tac ctt 12196 Leu Ala Ala Cys LeuArg Ser Ser Pro Lys Leu Lys Asn Lys Tyr Leu 205 210 215 ttt tgg ggc aggtac ttg aca caa ttc caa atg ttc cag ttt atg ctg 12244 Phe Trp Gly ArgTyr Leu Thr Gln Phe Gln Met Phe Gln Phe Met Leu 220 225 230 aac tta gtgcag gct tac tac gac atg aaa acg aat gcg cca tat cca 12292 Asn Leu ValGln Ala Tyr Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro 235 240 245 250 caatgg ctg atc aag att ttg ttc tac tac atg atc tcg ttg ctg ttt 12340 GlnTrp Leu Ile Lys Ile Leu Phe Tyr Tyr Met Ile Ser Leu Leu Phe 255 260 265ctt ttc ggc aat ttt tac gta caa aaa tac atc aaa ccc tct gac gga 12388Leu Phe Gly Asn Phe Tyr Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly 270 275280 aag caa aag gga gct aaa act gag tga tctagaaggc ctcctgcttt 12435 LysGln Lys Gly Ala Lys Thr Glu 285 290 aatgagatat gcgagacgcc tatgatcgcatgatatttgc tttcaattct gttgtgcacg 12495 ttgtaaaaaa cctgagcatg tgtagctcagatccttaccg ccggtttcgg ttcattctaa 12555 tgaatatatc acccgttact atcgtatttttatgaataat attctccgtt caatttactg 12615 attgtccgtc gagcaaattt acacattgccactaaacgtc taaacccttg taatttgttt 12675 ttgttttact atgtgtgtta tgtatttgatttgcgataaa tttttatatt tggtactaaa 12735 tttataacac cttttatgct aacgtttgccaacacttagc aatttgcaag ttgattaatt 12795 gattctaaat tatttttgtc ttctaaatacatatactaat caactggaaa tgtaaatatt 12855 tgctaatatt tctactatag gagaattaaagtgagtgaat atggtaccac aaggtttgga 12915 gatttaattg ttgcaatgct gcatggatggcatatacacc aaacattcaa taattcttga 12975 ggataataat ggtaccacac aagatttgaggtgcatgaac gtcacgtgga caaaaggttt 13035 agtaattttt caagacaaca atgttaccacacacaagttt tgaggtgcat gcatggatgc 13095 cctgtggaaa gtttaaaaat attttggaaatgatttgcat ggaagccatg tgtaaaacca 13155 tgacatccac ttggaggatg caataatgaagaaaactaca aatttacatg caactagtta 13215 tgcatgtagt ctatataatg aggattttgcaatactttca ttcatacaca ctcactaagt 13275 tttacacgat tataatttct tcatagccagcggatcc atg gta ttc gcg ggc ggt 13330 Met Val Phe Ala Gly Gly 295 ggactt cag cag ggc tct ctc gaa gaa aac atc gac gtc gag cac att 13378 GlyLeu Gln Gln Gly Ser Leu Glu Glu Asn Ile Asp Val Glu His Ile 300 305 310gcc agt atg tct ctc ttc agc gac ttc ttc agt tat gtg tct tca act 13426Ala Ser Met Ser Leu Phe Ser Asp Phe Phe Ser Tyr Val Ser Ser Thr 315 320325 gtt ggt tcg tgg agc gta cac agt ata caa cct ttg aag cgc ctg acg13474 Val Gly Ser Trp Ser Val His Ser Ile Gln Pro Leu Lys Arg Leu Thr330 335 340 agt aag aag cgt gtt tcg gaa agc gct gcc gtg caa tgt ata tcagct 13522 Ser Lys Lys Arg Val Ser Glu Ser Ala Ala Val Gln Cys Ile SerAla 345 350 355 360 gaa gtt cag aga aat tcg agt acc cag gga act gcg gaggca ctc gca 13570 Glu Val Gln Arg Asn Ser Ser Thr Gln Gly Thr Ala GluAla Leu Ala 365 370 375 gaa tca gtc gtg aag ccc acg aga cga agg tca tctcag tgg aag aag 13618 Glu Ser Val Val Lys Pro Thr Arg Arg Arg Ser SerGln Trp Lys Lys 380 385 390 tcg aca cac ccc cta tca gaa gta gca gta cacaac aag cca agc gat 13666 Ser Thr His Pro Leu Ser Glu Val Ala Val HisAsn Lys Pro Ser Asp 395 400 405 tgc tgg att gtt gta aaa aac aag gtg tatgat gtt tcc aat ttt gcg 13714 Cys Trp Ile Val Val Lys Asn Lys Val TyrAsp Val Ser Asn Phe Ala 410 415 420 gac gag cat ccc gga gga tca gtt attagt act tat ttt gga cga gac 13762 Asp Glu His Pro Gly Gly Ser Val IleSer Thr Tyr Phe Gly Arg Asp 425 430 435 440 ggc aca gat gtt ttc tct agtttt cat gca gct tct aca tgg aaa att 13810 Gly Thr Asp Val Phe Ser SerPhe His Ala Ala Ser Thr Trp Lys Ile 445 450 455 ctt caa gac ttt tac attggt gac gtg gag agg gtg gag ccg act cca 13858 Leu Gln Asp Phe Tyr IleGly Asp Val Glu Arg Val Glu Pro Thr Pro 460 465 470 gag ctg ctg aaa gatttc cga gaa atg aga gct ctt ttc ctg agg gag 13906 Glu Leu Leu Lys AspPhe Arg Glu Met Arg Ala Leu Phe Leu Arg Glu 475 480 485 caa ctt ttc aaaagt tcg aaa ttg tac tat gtt atg aag ctg ctc acg 13954 Gln Leu Phe LysSer Ser Lys Leu Tyr Tyr Val Met Lys Leu Leu Thr 490 495 500 aat gtt gctatt ttt gct gcg agc att gca ata ata tgt tgg agc aag 14002 Asn Val AlaIle Phe Ala Ala Ser Ile Ala Ile Ile Cys Trp Ser Lys 505 510 515 520 actatt tca gcg gtt ttg gct tca gct tgt atg atg gct ctg tgt ttc 14050 ThrIle Ser Ala Val Leu Ala Ser Ala Cys Met Met Ala Leu Cys Phe 525 530 535caa cag tgc gga tgg cta tcc cat gat ttt ctc cac aat cag gtg ttt 14098Gln Gln Cys Gly Trp Leu Ser His Asp Phe Leu His Asn Gln Val Phe 540 545550 gag aca cgc tgg ctt aat gaa gtt gtc ggg tat gtg atc ggc aac gcc14146 Glu Thr Arg Trp Leu Asn Glu Val Val Gly Tyr Val Ile Gly Asn Ala555 560 565 gtt ctg ggg ttt agt aca ggg tgg tgg aag gag aag cat aac cttcat 14194 Val Leu Gly Phe Ser Thr Gly Trp Trp Lys Glu Lys His Asn LeuHis 570 575 580 cat gct gct cca aat gaa tgc gat cag act tac caa cca attgat gaa 14242 His Ala Ala Pro Asn Glu Cys Asp Gln Thr Tyr Gln Pro IleAsp Glu 585 590 595 600 gat att gat act ctc ccc ctc att gcc tgg agc aaggac ata ctg gcc 14290 Asp Ile Asp Thr Leu Pro Leu Ile Ala Trp Ser LysAsp Ile Leu Ala 605 610 615 aca gtt gag aat aag aca ttc ttg cga atc ctccaa tac cag cat ctg 14338 Thr Val Glu Asn Lys Thr Phe Leu Arg Ile LeuGln Tyr Gln His Leu 620 625 630 ttc ttc atg ggt ctg tta ttt ttc gcc cgtggt agt tgg ctc ttt tgg 14386 Phe Phe Met Gly Leu Leu Phe Phe Ala ArgGly Ser Trp Leu Phe Trp 635 640 645 agc tgg aga tat acc tct aca gca gtgctc tca cct gtc gac agg ttg 14434 Ser Trp Arg Tyr Thr Ser Thr Ala ValLeu Ser Pro Val Asp Arg Leu 650 655 660 ttg gag aag gga act gtt ctg tttcac tac ttt tgg ttc gtc ggg aca 14482 Leu Glu Lys Gly Thr Val Leu PheHis Tyr Phe Trp Phe Val Gly Thr 665 670 675 680 gcg tgc tat ctt ctc cctggt tgg aag cca tta gta tgg atg gcg gtg 14530 Ala Cys Tyr Leu Leu ProGly Trp Lys Pro Leu Val Trp Met Ala Val 685 690 695 act gag ctc atg tccggc atg ctg ctg ggc ttt gta ttt gta ctt agc 14578 Thr Glu Leu Met SerGly Met Leu Leu Gly Phe Val Phe Val Leu Ser 700 705 710 cac aat ggg atggag gtt tat aat tcg tct aaa gaa ttc gtg agt gca 14626 His Asn Gly MetGlu Val Tyr Asn Ser Ser Lys Glu Phe Val Ser Ala 715 720 725 cag atc gtatcc aca cgg gat atc aaa gga aac ata ttc aac gac tgg 14674 Gln Ile ValSer Thr Arg Asp Ile Lys Gly Asn Ile Phe Asn Asp Trp 730 735 740 ttc actggt ggc ctt aac agg caa ata gag cat cat ctt ttc cca aca 14722 Phe ThrGly Gly Leu Asn Arg Gln Ile Glu His His Leu Phe Pro Thr 745 750 755 760atg ccc agg cat aat tta aac aaa ata gca cct aga gtg gag gtg ttc 14770Met Pro Arg His Asn Leu Asn Lys Ile Ala Pro Arg Val Glu Val Phe 765 770775 tgt aag aaa cac ggt ctg gtg tac gaa gac gta tct att gct acc ggc14818 Cys Lys Lys His Gly Leu Val Tyr Glu Asp Val Ser Ile Ala Thr Gly780 785 790 act tgc aag gtt ttg aaa gca ttg aag gaa gtc gcg gag gct gcggca 14866 Thr Cys Lys Val Leu Lys Ala Leu Lys Glu Val Ala Glu Ala AlaAla 795 800 805 gag cag cat gct acc acc agt taa gctagcgtta accctgctttaatgagatat 14920 Glu Gln His Ala Thr Thr Ser 810 815 gcgagacgcctatgatcgca tgatatttgc tttcaattct gttgtgcacg ttgtaaaaaa 14980 cctgagcatgtgtagctcag atccttaccg ccggtttcgg ttcattctaa tgaatatatc 15040 acccgttactatcgtatttt tatgaataat attctccgtt caatttactg attgtccgtc 15100 gacgaattcgagctcggcgc gcctctagag gatcgatgaa ttcagatcgg ctgagtggct 15160 ccttcaacgttgcggttctg tcagttccaa acgtaaaacg gcttgtcccg cgtcatcggc 15220 gggggtcataacgtgactcc cttaattctc cgctcatgat cagattgtcg tttcccgcct 15280 tcagtttaaactatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag 15340 cgtttattagaataatcgga tatttaaaag ggcgtgaaaa ggtttatcct tcgtccattt 15400 gtatgtgcatgccaaccaca gggttcccca 15430 26 290 PRT Unknown Plant expression vectorwith two promoter- terminator expression cassettes; Physcomitrellapatens elongase and desaturase are inserted 26 Met Glu Val Val Glu ArgPhe Tyr Gly Glu Leu Asp Gly Lys Val Ser 1 5 10 15 Gln Gly Val Asn AlaLeu Leu Gly Ser Phe Gly Val Glu Leu Thr Asp 20 25 30 Thr Pro Thr Thr LysGly Leu Pro Leu Val Asp Ser Pro Thr Pro Ile 35 40 45 Val Leu Gly Val SerVal Tyr Leu Thr Ile Val Ile Gly Gly Leu Leu 50 55 60 Trp Ile Lys Ala ArgAsp Leu Lys Pro Arg Ala Ser Glu Pro Phe Leu 65 70 75 80 Leu Gln Ala LeuVal Leu Val His Asn Leu Phe Cys Phe Ala Leu Ser 85 90 95 Leu Tyr Met CysVal Gly Ile Ala Tyr Gln Ala Ile Thr Trp Arg Tyr 100 105 110 Ser Leu TrpGly Asn Ala Tyr Asn Pro Lys His Lys Glu Met Ala Ile 115 120 125 Leu ValTyr Leu Phe Tyr Met Ser Lys Tyr Val Glu Phe Met Asp Thr 130 135 140 ValIle Met Ile Leu Lys Arg Ser Thr Arg Gln Ile Ser Phe Leu His 145 150 155160 Val Tyr His His Ser Ser Ile Ser Leu Ile Trp Trp Ala Ile Ala His 165170 175 His Ala Pro Gly Gly Glu Ala Tyr Trp Ser Ala Ala Leu Asn Ser Gly180 185 190 Val His Val Leu Met Tyr Ala Tyr Tyr Phe Leu Ala Ala Cys LeuArg 195 200 205 Ser Ser Pro Lys Leu Lys Asn Lys Tyr Leu Phe Trp Gly ArgTyr Leu 210 215 220 Thr Gln Phe Gln Met Phe Gln Phe Met Leu Asn Leu ValGln Ala Tyr 225 230 235 240 Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro GlnTrp Leu Ile Lys Ile 245 250 255 Leu Phe Tyr Tyr Met Ile Ser Leu Leu PheLeu Phe Gly Asn Phe Tyr 260 265 270 Val Gln Lys Tyr Ile Lys Pro Ser AspGly Lys Gln Lys Gly Ala Lys 275 280 285 Thr Glu 290 27 525 PRT UnknownPlant expression vector with two promoter- terminator expressioncassettes; Physcomitrella patens elongase and desaturase are inserted 27Met Val Phe Ala Gly Gly Gly Leu Gln Gln Gly Ser Leu Glu Glu Asn 1 5 1015 Ile Asp Val Glu His Ile Ala Ser Met Ser Leu Phe Ser Asp Phe Phe 20 2530 Ser Tyr Val Ser Ser Thr Val Gly Ser Trp Ser Val His Ser Ile Gln 35 4045 Pro Leu Lys Arg Leu Thr Ser Lys Lys Arg Val Ser Glu Ser Ala Ala 50 5560 Val Gln Cys Ile Ser Ala Glu Val Gln Arg Asn Ser Ser Thr Gln Gly 65 7075 80 Thr Ala Glu Ala Leu Ala Glu Ser Val Val Lys Pro Thr Arg Arg Arg 8590 95 Ser Ser Gln Trp Lys Lys Ser Thr His Pro Leu Ser Glu Val Ala Val100 105 110 His Asn Lys Pro Ser Asp Cys Trp Ile Val Val Lys Asn Lys ValTyr 115 120 125 Asp Val Ser Asn Phe Ala Asp Glu His Pro Gly Gly Ser ValIle Ser 130 135 140 Thr Tyr Phe Gly Arg Asp Gly Thr Asp Val Phe Ser SerPhe His Ala 145 150 155 160 Ala Ser Thr Trp Lys Ile Leu Gln Asp Phe TyrIle Gly Asp Val Glu 165 170 175 Arg Val Glu Pro Thr Pro Glu Leu Leu LysAsp Phe Arg Glu Met Arg 180 185 190 Ala Leu Phe Leu Arg Glu Gln Leu PheLys Ser Ser Lys Leu Tyr Tyr 195 200 205 Val Met Lys Leu Leu Thr Asn ValAla Ile Phe Ala Ala Ser Ile Ala 210 215 220 Ile Ile Cys Trp Ser Lys ThrIle Ser Ala Val Leu Ala Ser Ala Cys 225 230 235 240 Met Met Ala Leu CysPhe Gln Gln Cys Gly Trp Leu Ser His Asp Phe 245 250 255 Leu His Asn GlnVal Phe Glu Thr Arg Trp Leu Asn Glu Val Val Gly 260 265 270 Tyr Val IleGly Asn Ala Val Leu Gly Phe Ser Thr Gly Trp Trp Lys 275 280 285 Glu LysHis Asn Leu His His Ala Ala Pro Asn Glu Cys Asp Gln Thr 290 295 300 TyrGln Pro Ile Asp Glu Asp Ile Asp Thr Leu Pro Leu Ile Ala Trp 305 310 315320 Ser Lys Asp Ile Leu Ala Thr Val Glu Asn Lys Thr Phe Leu Arg Ile 325330 335 Leu Gln Tyr Gln His Leu Phe Phe Met Gly Leu Leu Phe Phe Ala Arg340 345 350 Gly Ser Trp Leu Phe Trp Ser Trp Arg Tyr Thr Ser Thr Ala ValLeu 355 360 365 Ser Pro Val Asp Arg Leu Leu Glu Lys Gly Thr Val Leu PheHis Tyr 370 375 380 Phe Trp Phe Val Gly Thr Ala Cys Tyr Leu Leu Pro GlyTrp Lys Pro 385 390 395 400 Leu Val Trp Met Ala Val Thr Glu Leu Met SerGly Met Leu Leu Gly 405 410 415 Phe Val Phe Val Leu Ser His Asn Gly MetGlu Val Tyr Asn Ser Ser 420 425 430 Lys Glu Phe Val Ser Ala Gln Ile ValSer Thr Arg Asp Ile Lys Gly 435 440 445 Asn Ile Phe Asn Asp Trp Phe ThrGly Gly Leu Asn Arg Gln Ile Glu 450 455 460 His His Leu Phe Pro Thr MetPro Arg His Asn Leu Asn Lys Ile Ala 465 470 475 480 Pro Arg Val Glu ValPhe Cys Lys Lys His Gly Leu Val Tyr Glu Asp 485 490 495 Val Ser Ile AlaThr Gly Thr Cys Lys Val Leu Lys Ala Leu Lys Glu 500 505 510 Val Ala GluAla Ala Ala Glu Gln His Ala Thr Thr Ser 515 520 525 28 17752 DNA UnknownPlant expression vector with 3 promoter- terminator expression cassetteswith Physcomitrella elongase + desaturase + Phaeodactylum desaturaseinserted 28 gatctggcgc cggccagcga gacgagcaag attggccgcc gcccgaaacgatccgacagc 60 gcgcccagca caggtgcgca ggcaaattgc accaacgcat acagcgccagcagaatgcca 120 tagtgggcgg tgacgtcgtt cgagtgaacc agatcgcgca ggaggcccggcagcaccggc 180 ataatcaggc cgatgccgac agcgtcgagc gcgacagtgc tcagaattacgatcaggggt 240 atgttgggtt tcacgtctgg cctccggacc agcctccgct ggtccgattgaacgcgcgga 300 ttctttatca ctgataagtt ggtggacata ttatgtttat cagtgataaagtgtcaagca 360 tgacaaagtt gcagccgaat acagtgatcc gtgccgccct ggacctgttgaacgaggtcg 420 gcgtagacgg tctgacgaca cgcaaactgg cggaacggtt gggggttcagcagccggcgc 480 tttactggca cttcaggaac aagcgggcgc tgctcgacgc actggccgaagccatgctgg 540 cggagaatca tacgcattcg gtgccgagag ccgacgacga ctggcgctcatttctgatcg 600 ggaatgcccg cagcttcagg caggcgctgc tcgcctaccg cgatggcgcgcgcatccatg 660 ccggcacgcg accgggcgca ccgcagatgg aaacggccga cgcgcagcttcgcttcctct 720 gcgaggcggg tttttcggcc ggggacgccg tcaatgcgct gatgacaatcagctacttca 780 ctgttggggc cgtgcttgag gagcaggccg gcgacagcga tgccggcgagcgcggcggca 840 ccgttgaaca ggctccgctc tcgccgctgt tgcgggccgc gatagacgccttcgacgaag 900 ccggtccgga cgcagcgttc gagcagggac tcgcggtgat tgtcgatggattggcgaaaa 960 ggaggctcgt tgtcaggaac gttgaaggac cgagaaaggg tgacgattgatcaggaccgc 1020 tgccggagcg caacccactc actacagcag agccatgtag acaacatcccctcccccttt 1080 ccaccgcgtc agacgcccgt agcagcccgc tacgggcttt ttcatgccctgccctagcgt 1140 ccaagcctca cggccgcgct cggcctctct ggcggccttc tggcgctcttccgcttcctc 1200 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcagctcactcaaa 1260 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaacatgtgagcaaa 1320 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttttccataggct 1380 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggcgaaacccgac 1440 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgctctcctgttcc 1500 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcgtggcgctttt 1560 ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatatccatcctttt 1620 tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttggtgtatccaac 1680 ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgttccttcttca 1740 ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcgaggctggccgg 1800 ctaccgccgg cgtaacagat gagggcaagc ggatggctga tgaaaccaagccaaccagga 1860 agggcagccc acctatcaag gtgtactgcc ttccagacga acgaagagcgattgaggaaa 1920 aggcggcggc ggccggcatg agcctgtcgg cctacctgct ggccgtcggccagggctaca 1980 aaatcacggg cgtcgtggac tatgagcacg tccgcgagct ggcccgcatcaatggcgacc 2040 tgggccgcct gggcggcctg ctgaaactct ggctcaccga cgacccgcgcacggcgcggt 2100 tcggtgatgc cacgatcctc gccctgctgg cgaagatcga agagaagcaggacgagcttg 2160 gcaaggtcat gatgggcgtg gtccgcccga gggcagagcc atgacttttttagccgctaa 2220 aacggccggg gggtgcgcgt gattgccaag cacgtcccca tgcgctccatcaagaagagc 2280 gacttcgcgg agctggtgaa gtacatcacc gacgagcaag gcaagaccgagcgcctttgc 2340 gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatggccctgcaaa 2400 cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggcgttgtggata 2460 cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgacacttgaggggc 2520 cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggccggcgacgtg 2580 gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtttcccacagat 2640 gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggggcgcgactac 2700 tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgacagatgaggggc 2760 gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcatttgcaagggttt 2820 ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaaccaatatttat 2880 aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgccgaagggggg 2940 tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatccccccaggggc 3000 tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agcgctggcagtccttgcca 3060 ttgccgggat cggggcagta acgggatggg cgatcagccc gagcgcgacgcccggaagca 3120 ttgacgtgcc gcaggtgctg gcatcgacat tcagcgacca ggtgccgggcagtgagggcg 3180 gcggcctggg tggcggcctg cccttcactt cggccgtcgg ggcattcacggacttcatgg 3240 cggggccggc aatttttacc ttgggcattc ttggcatagt ggtcgcgggtgccgtgctcg 3300 tgttcggggg tgcgataaac ccagcgaacc atttgaggtg ataggtaagattataccgag 3360 gtatgaaaac gagaattgga cctttacaga attactctat gaagcgccatatttaaaaag 3420 ctaccaagac gaagaggatg aagaggatga ggaggcagat tgccttgaatatattgacaa 3480 tactgataag ataatatatc ttttatatag aagatatcgc cgtatgtaaggatttcaggg 3540 ggcaaggcat aggcagcgcg cttatcaata tatctataga atgggcaaagcataaaaact 3600 tgcatggact aatgcttgaa acccaggaca ataaccttat agcttgtaaattctatcata 3660 attgggtaat gactccaact tattgatagt gttttatgtt cagataatgcccgatgactt 3720 tgtcatgcag ctccaccgat tttgagaacg acagcgactt ccgtcccagccgtgccaggt 3780 gctgcctcag attcaggtta tgccgctcaa ttcgctgcgt atatcgcttgctgattacgt 3840 gcagctttcc cttcaggcgg gattcataca gcggccagcc atccgtcatccatatcacca 3900 cgtcaaaggg tgacagcagg ctcataagac gccccagcgt cgccatagtgcgttcaccga 3960 atacgtgcgc aacaaccgtc ttccggagac tgtcatacgc gtaaaacagccagcgctggc 4020 gcgatttagc cccgacatag ccccactgtt cgtccatttc cgcgcagacgatgacgtcac 4080 tgcccggctg tatgcgcgag gttaccgact gcggcctgag ttttttaagtgacgtaaaat 4140 cgtgttgagg ccaacgccca taatgcgggc tgttgcccgg catccaacgccattcatggc 4200 catatcaatg attttctggt gcgtaccggg ttgagaagcg gtgtaagtgaactgcagttg 4260 ccatgtttta cggcagtgag agcagagata gcgctgatgt ccggcggtgcttttgccgtt 4320 acgcaccacc ccgtcagtag ctgaacagga gggacagctg atagacacagaagccactgg 4380 agcacctcaa aaacaccatc atacactaaa tcagtaagtt ggcagcatcacccataattg 4440 tggtttcaaa atcggctccg tcgatactat gttatacgcc aactttgaaaacaactttga 4500 aaaagctgtt ttctggtatt taaggtttta gaatgcaagg aacagtgaattggagttcgt 4560 cttgttataa ttagcttctt ggggtatctt taaatactgt agaaaagaggaaggaaataa 4620 taaatggcta aaatgagaat atcaccggaa ttgaaaaaac tgatcgaaaaataccgctgc 4680 gtaaaagata cggaaggaat gtctcctgct aaggtatata agctggtgggagaaaatgaa 4740 aacctatatt taaaaatgac ggacagccgg tataaaggga ccacctatgatgtggaacgg 4800 gaaaaggaca tgatgctatg gctggaagga aagctgcctg ttccaaaggtcctgcacttt 4860 gaacggcatg atggctggag caatctgctc atgagtgagg ccgatggcgtcctttgctcg 4920 gaagagtatg aagatgaaca aagccctgaa aagattatcg agctgtatgcggagtgcatc 4980 aggctctttc actccatcga catatcggat tgtccctata cgaatagcttagacagccgc 5040 ttagccgaat tggattactt actgaataac gatctggccg atgtggattgcgaaaactgg 5100 gaagaagaca ctccatttaa agatccgcgc gagctgtatg attttttaaagacggaaaag 5160 cccgaagagg aacttgtctt ttcccacggc gacctgggag acagcaacatctttgtgaaa 5220 gatggcaaag taagtggctt tattgatctt gggagaagcg gcagggcggacaagtggtat 5280 gacattgcct tctgcgtccg gtcgatcagg gaggatatcg gggaagaacagtatgtcgag 5340 ctattttttg acttactggg gatcaagcct gattgggaga aaataaaatattatatttta 5400 ctggatgaat tgttttagta cctagatgtg gcgcaacgat gccggcgacaagcaggagcg 5460 caccgacttc ttccgcatca agtgttttgg ctctcaggcc gaggcccacggcaagtattt 5520 gggcaagggg tcgctggtat tcgtgcaggg caagattcgg aataccaagtacgagaagga 5580 cggccagacg gtctacggga ccgacttcat tgccgataag gtggattatctggacaccaa 5640 ggcaccaggc gggtcaaatc aggaataagg gcacattgcc ccggcgtgagtcggggcaat 5700 cccgcaagga gggtgaatga atcggacgtt tgaccggaag gcatacaggcaagaactgat 5760 cgacgcgggg ttttccgccg aggatgccga aaccatcgca agccgcaccgtcatgcgtgc 5820 gccccgcgaa accttccagt ccgtcggctc gatggtccag caagctacggccaagatcga 5880 gcgcgacagc gtgcaactgg ctccccctgc cctgcccgcg ccatcggccgccgtggagcg 5940 ttcgcgtcgt ctcgaacagg aggcggcagg tttggcgaag tcgatgaccatcgacacgcg 6000 aggaactatg acgaccaaga agcgaaaaac cgccggcgag gacctggcaaaacaggtcag 6060 cgaggccaag caggccgcgt tgctgaaaca cacgaagcag cagatcaaggaaatgcagct 6120 ttccttgttc gatattgcgc cgtggccgga cacgatgcga gcgatgccaaacgacacggc 6180 ccgctctgcc ctgttcacca cgcgcaacaa gaaaatcccg cgcgaggcgctgcaaaacaa 6240 ggtcattttc cacgtcaaca aggacgtgaa gatcacctac accggcgtcgagctgcgggc 6300 cgacgatgac gaactggtgt ggcagcaggt gttggagtac gcgaagcgcacccctatcgg 6360 cgagccgatc accttcacgt tctacgagct ttgccaggac ctgggctggtcgatcaatgg 6420 ccggtattac acgaaggccg aggaatgcct gtcgcgccta caggcgacggcgatgggctt 6480 cacgtccgac cgcgttgggc acctggaatc ggtgtcgctg ctgcaccgcttccgcgtcct 6540 ggaccgtggc aagaaaacgt cccgttgcca ggtcctgatc gacgaggaaatcgtcgtgct 6600 gtttgctggc gaccactaca cgaaattcat atgggagaag taccgcaagctgtcgccgac 6660 ggcccgacgg atgttcgact atttcagctc gcaccgggag ccgtacccgctcaagctgga 6720 aaccttccgc ctcatgtgcg gatcggattc cacccgcgtg aagaagtggcgcgagcaggt 6780 cggcgaagcc tgcgaagagt tgcgaggcag cggcctggtg gaacacgcctgggtcaatga 6840 tgacctggtg cattgcaaac gctagggcct tgtggggtca gttccggctgggggttcagc 6900 agccagcgct ttactggcat ttcaggaaca agcgggcact gctcgacgcacttgcttcgc 6960 tcagtatcgc tcgggacgca cggcgcgctc tacgaactgc cgataaacagaggattaaaa 7020 ttgacaattg tgattaaggc tcagattcga cggcttggag cggccgacgtgcaggatttc 7080 cgcgagatcc gattgtcggc cctgaagaaa gctccagaga tgttcgggtccgtttacgag 7140 cacgaggaga aaaagcccat ggaggcgttc gctgaacggt tgcgagatgccgtggcattc 7200 ggcgcctaca tcgacggcga gatcattggg ctgtcggtct tcaaacaggaggacggcccc 7260 aaggacgctc acaaggcgca tctgtccggc gttttcgtgg agcccgaacagcgaggccga 7320 ggggtcgccg gtatgctgct gcgggcgttg ccggcgggtt tattgctcgtgatgatcgtc 7380 cgacagattc caacgggaat ctggtggatg cgcatcttca tcctcggcgcacttaatatt 7440 tcgctattct ggagcttgtt gtttatttcg gtctaccgcc tgccgggcggggtcgcggcg 7500 acggtaggcg ctgtgcagcc gctgatggtc gtgttcatct ctgccgctctgctaggtagc 7560 ccgatacgat tgatggcggt cctgggggct atttgcggaa ctgcgggcgtggcgctgttg 7620 gtgttgacac caaacgcagc gctagatcct gtcggcgtcg cagcgggcctggcgggggcg 7680 gtttccatgg cgttcggaac cgtgctgacc cgcaagtggc aacctcccgtgcctctgctc 7740 acctttaccg cctggcaact ggcggccgga ggacttctgc tcgttccagtagctttagtg 7800 tttgatccgc caatcccgat gcctacagga accaatgttc tcggcctggcgtggctcggc 7860 ctgatcggag cgggtttaac ctacttcctt tggttccggg ggatctcgcgactcgaacct 7920 acagttgttt ccttactggg ctttctcagc cccagatctg gggtcgatcagccggggatg 7980 catcaggccg acagtcggaa cttcgggtcc ccgacctgta ccattcggtgagcaatggat 8040 aggggagttg atatcgtcaa cgttcacttc taaagaaata gcgccactcagcttcctcag 8100 cggctttatc cagcgatttc ctattatgtc ggcatagttc tcaagatcgacagcctgtca 8160 cggttaagcg agaaatgaat aagaaggctg ataattcgga tctctgcgagggagatgata 8220 tttgatcaca ggcagcaacg ctctgtcatc gttacaatca acatgctaccctccgcgaga 8280 tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt ccgaatagcatcggtaacat 8340 gagcaaagtc tgccgcctta caacggctct cccgctgacg ccgtcccggactgatgggct 8400 gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg gggagctgttggctggctgg 8460 tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaataacacattgcg 8520 gacgttttta atgtactggg gtggtttttc ttttcaccag tgagacgggcaacagctgat 8580 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctggtttgcccca 8640 gcaggcgaaa atcctgtttg atggtggttc cgaaatcggc aaaatcccttataaatcaaa 8700 agaatagccc gagatagggt tgagtgttgt tccagtttgg aacaagagtccactattaaa 8760 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatggcccactacg 8820 tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcactaaatcggaa 8880 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacgtggcgagaaa 8940 ggaagggaag aaagcgaaag gagcgggcgc cattcaggct gcgcaactgttgggaagggc 9000 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgtgctgcaaggc 9060 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacgacggccagtg 9120 aattaattcc catcttgaaa gaaatatagt ttaaatattt attgataaaataacaagtca 9180 ggtattatag tccaagcaaa aacataaatt tattgatgca agtttaaattcagaaatatt 9240 tcaataactg attatatcag ctggtacatt gccgtagatg aaagactgagtgcgatatta 9300 tgtgtaatac ataaattgat gatatagcta gcttagctca tcgggggatccgtcgaagct 9360 agcttgggtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgatgcgctgcgaa 9420 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgccgccaagctct 9480 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccacacccagccgg 9540 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcggcaagcaggca 9600 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgagcctggcgaac 9660 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatcgacaagaccg 9720 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtcgaatgggcag 9780 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatggatactttctcg 9840 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaatagcagccag 9900 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcccgtcgtggcc 9960 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccggacaggtcggtc 10020 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggcatcagagcag 10080 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagcggccggagaa 10140 cctgcgtgca atccatcttg ttcaatccaa gctcccatgg gccctcgactagagtcgaga 10200 tctggattga gagtgaatat gagactctaa ttggataccg aggggaatttatggaacgtc 10260 agtggagcat ttttgacaag aaatatttgc tagctgatag tgaccttaggcgacttttga 10320 acgcgcaata atggtttctg acgtatgtgc ttagctcatt aaactccagaaacccgcggc 10380 tgagtggctc cttcaacgtt gcggttctgt cagttccaaa cgtaaaacggcttgtcccgc 10440 gtcatcggcg ggggtcataa cgtgactccc ttaattctcc gctcatgatcttgatcccct 10500 gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagggcttcccaac 10560 cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccataaaaccgccca 10620 gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttgcgcttgcgtt 10680 ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcaccgtttctgcgg 10740 actggctttc tacgtgttcc gcttccttta gcagcccttg cgccctgagtgcttgcggca 10800 gcgtgaagct tgcatgcctg caggtcgacg gcgcgccgag ctcctcgagcaaatttacac 10860 attgccacta aacgtctaaa cccttgtaat ttgtttttgt tttactatgtgtgttatgta 10920 tttgatttgc gataaatttt tatatttggt actaaattta taacaccttttatgctaacg 10980 tttgccaaca cttagcaatt tgcaagttga ttaattgatt ctaaattatttttgtcttct 11040 aaatacatat actaatcaac tggaaatgta aatatttgct aatatttctactataggaga 11100 attaaagtga gtgaatatgg taccacaagg tttggagatt taattgttgcaatgctgcat 11160 ggatggcata tacaccaaac attcaataat tcttgaggat aataatggtaccacacaaga 11220 tttgaggtgc atgaacgtca cgtggacaaa aggtttagta atttttcaagacaacaatgt 11280 taccacacac aagttttgag gtgcatgcat ggatgccctg tggaaagtttaaaaatattt 11340 tggaaatgat ttgcatggaa gccatgtgta aaaccatgac atccacttggaggatgcaat 11400 aatgaagaaa actacaaatt tacatgcaac tagttatgca tgtagtctatataatgagga 11460 ttttgcaata ctttcattca tacacactca ctaagtttta cacgattataatttcttcat 11520 agccagccca ccgcggtgga aa atg gag gtc gtg gag aga ttctac ggt gag 11572 Met Glu Val Val Glu Arg Phe Tyr Gly Glu 1 5 10 ttg gatggg aag gtc tcg cag ggc gtg aat gca ttg ctg ggt agt ttt 11620 Leu AspGly Lys Val Ser Gln Gly Val Asn Ala Leu Leu Gly Ser Phe 15 20 25 ggg gtggag ttg acg gat acg ccc act acc aaa ggc ttg ccc ctc gtt 11668 Gly ValGlu Leu Thr Asp Thr Pro Thr Thr Lys Gly Leu Pro Leu Val 30 35 40 gac agtccc aca ccc atc gtc ctc ggt gtt tct gta tac ttg act att 11716 Asp SerPro Thr Pro Ile Val Leu Gly Val Ser Val Tyr Leu Thr Ile 45 50 55 gtc attgga ggg ctt ttg tgg ata aag gcc agg gat ctg aaa ccg cgc 11764 Val IleGly Gly Leu Leu Trp Ile Lys Ala Arg Asp Leu Lys Pro Arg 60 65 70 gcc tcggag cca ttt ttg ctc caa gct ttg gtg ctt gtg cac aac ctg 11812 Ala SerGlu Pro Phe Leu Leu Gln Ala Leu Val Leu Val His Asn Leu 75 80 85 90 ttctgt ttt gcg ctc agt ctg tat atg tgc gtg ggc atc gct tat cag 11860 PheCys Phe Ala Leu Ser Leu Tyr Met Cys Val Gly Ile Ala Tyr Gln 95 100 105gct att acc tgg cgg tac tct ctc tgg ggc aat gca tac aat cct aaa 11908Ala Ile Thr Trp Arg Tyr Ser Leu Trp Gly Asn Ala Tyr Asn Pro Lys 110 115120 cat aaa gag atg gcg att ctg gta tac ttg ttc tac atg tct aag tac11956 His Lys Glu Met Ala Ile Leu Val Tyr Leu Phe Tyr Met Ser Lys Tyr125 130 135 gtg gaa ttc atg gat acc gtt atc atg ata ctg aag cgc agc accagg 12004 Val Glu Phe Met Asp Thr Val Ile Met Ile Leu Lys Arg Ser ThrArg 140 145 150 caa ata agc ttc ctc cac gtt tat cat cat tct tca att tccctc att 12052 Gln Ile Ser Phe Leu His Val Tyr His His Ser Ser Ile SerLeu Ile 155 160 165 170 tgg tgg gct att gct cat cac gct cct ggc ggt gaagca tat tgg tct 12100 Trp Trp Ala Ile Ala His His Ala Pro Gly Gly GluAla Tyr Trp Ser 175 180 185 gcg gct ctg aac tca gga gtg cat gtt ctc atgtat gcg tat tac ttc 12148 Ala Ala Leu Asn Ser Gly Val His Val Leu MetTyr Ala Tyr Tyr Phe 190 195 200 ttg gct gcc tgc ctt cga agt agc cca aagtta aaa aat aag tac ctt 12196 Leu Ala Ala Cys Leu Arg Ser Ser Pro LysLeu Lys Asn Lys Tyr Leu 205 210 215 ttt tgg ggc agg tac ttg aca caa ttccaa atg ttc cag ttt atg ctg 12244 Phe Trp Gly Arg Tyr Leu Thr Gln PheGln Met Phe Gln Phe Met Leu 220 225 230 aac tta gtg cag gct tac tac gacatg aaa acg aat gcg cca tat cca 12292 Asn Leu Val Gln Ala Tyr Tyr AspMet Lys Thr Asn Ala Pro Tyr Pro 235 240 245 250 caa tgg ctg atc aag attttg ttc tac tac atg atc tcg ttg ctg ttt 12340 Gln Trp Leu Ile Lys IleLeu Phe Tyr Tyr Met Ile Ser Leu Leu Phe 255 260 265 ctt ttc ggc aat ttttac gta caa aaa tac atc aaa ccc tct gac gga 12388 Leu Phe Gly Asn PheTyr Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly 270 275 280 aag caa aag ggagct aaa act gag tga tctagaaggc ctcctgcttt 12435 Lys Gln Lys Gly Ala LysThr Glu 285 290 aatgagatat gcgagacgcc tatgatcgca tgatatttgc tttcaattctgttgtgcacg 12495 ttgtaaaaaa cctgagcatg tgtagctcag atccttaccg ccggtttcggttcattctaa 12555 tgaatatatc acccgttact atcgtatttt tatgaataat attctccgttcaatttactg 12615 attgtccgtc gagcaaattt acacattgcc actaaacgtc taaacccttgtaatttgttt 12675 ttgttttact atgtgtgtta tgtatttgat ttgcgataaa tttttatatttggtactaaa 12735 tttataacac cttttatgct aacgtttgcc aacacttagc aatttgcaagttgattaatt 12795 gattctaaat tatttttgtc ttctaaatac atatactaat caactggaaatgtaaatatt 12855 tgctaatatt tctactatag gagaattaaa gtgagtgaat atggtaccacaaggtttgga 12915 gatttaattg ttgcaatgct gcatggatgg catatacacc aaacattcaataattcttga 12975 ggataataat ggtaccacac aagatttgag gtgcatgaac gtcacgtggacaaaaggttt 13035 agtaattttt caagacaaca atgttaccac acacaagttt tgaggtgcatgcatggatgc 13095 cctgtggaaa gtttaaaaat attttggaaa tgatttgcat ggaagccatgtgtaaaacca 13155 tgacatccac ttggaggatg caataatgaa gaaaactaca aatttacatgcaactagtta 13215 tgcatgtagt ctatataatg aggattttgc aatactttca ttcatacacactcactaagt 13275 tttacacgat tataatttct tcatagccag cggatcc atg gta ttcgcg ggc ggt 13330 Met Val Phe Ala Gly Gly 295 gga ctt cag cag ggc tctctc gaa gaa aac atc gac gtc gag cac att 13378 Gly Leu Gln Gln Gly SerLeu Glu Glu Asn Ile Asp Val Glu His Ile 300 305 310 gcc agt atg tct ctcttc agc gac ttc ttc agt tat gtg tct tca act 13426 Ala Ser Met Ser LeuPhe Ser Asp Phe Phe Ser Tyr Val Ser Ser Thr 315 320 325 gtt ggt tcg tggagc gta cac agt ata caa cct ttg aag cgc ctg acg 13474 Val Gly Ser TrpSer Val His Ser Ile Gln Pro Leu Lys Arg Leu Thr 330 335 340 agt aag aagcgt gtt tcg gaa agc gct gcc gtg caa tgt ata tca gct 13522 Ser Lys LysArg Val Ser Glu Ser Ala Ala Val Gln Cys Ile Ser Ala 345 350 355 360 gaagtt cag aga aat tcg agt acc cag gga act gcg gag gca ctc gca 13570 GluVal Gln Arg Asn Ser Ser Thr Gln Gly Thr Ala Glu Ala Leu Ala 365 370 375gaa tca gtc gtg aag ccc acg aga cga agg tca tct cag tgg aag aag 13618Glu Ser Val Val Lys Pro Thr Arg Arg Arg Ser Ser Gln Trp Lys Lys 380 385390 tcg aca cac ccc cta tca gaa gta gca gta cac aac aag cca agc gat13666 Ser Thr His Pro Leu Ser Glu Val Ala Val His Asn Lys Pro Ser Asp395 400 405 tgc tgg att gtt gta aaa aac aag gtg tat gat gtt tcc aat tttgcg 13714 Cys Trp Ile Val Val Lys Asn Lys Val Tyr Asp Val Ser Asn PheAla 410 415 420 gac gag cat ccc gga gga tca gtt att agt act tat ttt ggacga gac 13762 Asp Glu His Pro Gly Gly Ser Val Ile Ser Thr Tyr Phe GlyArg Asp 425 430 435 440 ggc aca gat gtt ttc tct agt ttt cat gca gct tctaca tgg aaa att 13810 Gly Thr Asp Val Phe Ser Ser Phe His Ala Ala SerThr Trp Lys Ile 445 450 455 ctt caa gac ttt tac att ggt gac gtg gag agggtg gag ccg act cca 13858 Leu Gln Asp Phe Tyr Ile Gly Asp Val Glu ArgVal Glu Pro Thr Pro 460 465 470 gag ctg ctg aaa gat ttc cga gaa atg agagct ctt ttc ctg agg gag 13906 Glu Leu Leu Lys Asp Phe Arg Glu Met ArgAla Leu Phe Leu Arg Glu 475 480 485 caa ctt ttc aaa agt tcg aaa ttg tactat gtt atg aag ctg ctc acg 13954 Gln Leu Phe Lys Ser Ser Lys Leu TyrTyr Val Met Lys Leu Leu Thr 490 495 500 aat gtt gct att ttt gct gcg agcatt gca ata ata tgt tgg agc aag 14002 Asn Val Ala Ile Phe Ala Ala SerIle Ala Ile Ile Cys Trp Ser Lys 505 510 515 520 act att tca gcg gtt ttggct tca gct tgt atg atg gct ctg tgt ttc 14050 Thr Ile Ser Ala Val LeuAla Ser Ala Cys Met Met Ala Leu Cys Phe 525 530 535 caa cag tgc gga tggcta tcc cat gat ttt ctc cac aat cag gtg ttt 14098 Gln Gln Cys Gly TrpLeu Ser His Asp Phe Leu His Asn Gln Val Phe 540 545 550 gag aca cgc tggctt aat gaa gtt gtc ggg tat gtg atc ggc aac gcc 14146 Glu Thr Arg TrpLeu Asn Glu Val Val Gly Tyr Val Ile Gly Asn Ala 555 560 565 gtt ctg gggttt agt aca ggg tgg tgg aag gag aag cat aac ctt cat 14194 Val Leu GlyPhe Ser Thr Gly Trp Trp Lys Glu Lys His Asn Leu His 570 575 580 cat gctgct cca aat gaa tgc gat cag act tac caa cca att gat gaa 14242 His AlaAla Pro Asn Glu Cys Asp Gln Thr Tyr Gln Pro Ile Asp Glu 585 590 595 600gat att gat act ctc ccc ctc att gcc tgg agc aag gac ata ctg gcc 14290Asp Ile Asp Thr Leu Pro Leu Ile Ala Trp Ser Lys Asp Ile Leu Ala 605 610615 aca gtt gag aat aag aca ttc ttg cga atc ctc caa tac cag cat ctg14338 Thr Val Glu Asn Lys Thr Phe Leu Arg Ile Leu Gln Tyr Gln His Leu620 625 630 ttc ttc atg ggt ctg tta ttt ttc gcc cgt ggt agt tgg ctc ttttgg 14386 Phe Phe Met Gly Leu Leu Phe Phe Ala Arg Gly Ser Trp Leu PheTrp 635 640 645 agc tgg aga tat acc tct aca gca gtg ctc tca cct gtc gacagg ttg 14434 Ser Trp Arg Tyr Thr Ser Thr Ala Val Leu Ser Pro Val AspArg Leu 650 655 660 ttg gag aag gga act gtt ctg ttt cac tac ttt tgg ttcgtc ggg aca 14482 Leu Glu Lys Gly Thr Val Leu Phe His Tyr Phe Trp PheVal Gly Thr 665 670 675 680 gcg tgc tat ctt ctc cct ggt tgg aag cca ttagta tgg atg gcg gtg 14530 Ala Cys Tyr Leu Leu Pro Gly Trp Lys Pro LeuVal Trp Met Ala Val 685 690 695 act gag ctc atg tcc ggc atg ctg ctg ggcttt gta ttt gta ctt agc 14578 Thr Glu Leu Met Ser Gly Met Leu Leu GlyPhe Val Phe Val Leu Ser 700 705 710 cac aat ggg atg gag gtt tat aat tcgtct aaa gaa ttc gtg agt gca 14626 His Asn Gly Met Glu Val Tyr Asn SerSer Lys Glu Phe Val Ser Ala 715 720 725 cag atc gta tcc aca cgg gat atcaaa gga aac ata ttc aac gac tgg 14674 Gln Ile Val Ser Thr Arg Asp IleLys Gly Asn Ile Phe Asn Asp Trp 730 735 740 ttc act ggt ggc ctt aac aggcaa ata gag cat cat ctt ttc cca aca 14722 Phe Thr Gly Gly Leu Asn ArgGln Ile Glu His His Leu Phe Pro Thr 745 750 755 760 atg ccc agg cat aattta aac aaa ata gca cct aga gtg gag gtg ttc 14770 Met Pro Arg His AsnLeu Asn Lys Ile Ala Pro Arg Val Glu Val Phe 765 770 775 tgt aag aaa cacggt ctg gtg tac gaa gac gta tct att gct acc ggc 14818 Cys Lys Lys HisGly Leu Val Tyr Glu Asp Val Ser Ile Ala Thr Gly 780 785 790 act tgc aaggtt ttg aaa gca ttg aag gaa gtc gcg gag gct gcg gca 14866 Thr Cys LysVal Leu Lys Ala Leu Lys Glu Val Ala Glu Ala Ala Ala 795 800 805 gag cagcat gct acc acc agt taa gctagcgtta accctgcttt aatgagatat 14920 Glu GlnHis Ala Thr Thr Ser 810 815 gcgagacgcc tatgatcgca tgatatttgc tttcaattctgttgtgcacg ttgtaaaaaa 14980 cctgagcatg tgtagctcag atccttaccg ccggtttcggttcattctaa tgaatatatc 15040 acccgttact atcgtatttt tatgaataat attctccgttcaatttactg attgtccgtc 15100 gagcaaattt acacattgcc actaaacgtc taaacccttgtaatttgttt ttgttttact 15160 atgtgtgtta tgtatttgat ttgcgataaa tttttatatttggtactaaa tttataacac 15220 cttttatgct aacgtttgcc aacacttagc aatttgcaagttgattaatt gattctaaat 15280 tatttttgtc ttctaaatac atatactaat caactggaaatgtaaatatt tgctaatatt 15340 tctactatag gagaattaaa gtgagtgaat atggtaccacaaggtttgga gatttaattg 15400 ttgcaatgct gcatggatgg catatacacc aaacattcaataattcttga ggataataat 15460 ggtaccacac aagatttgag gtgcatgaac gtcacgtggacaaaaggttt agtaattttt 15520 caagacaaca atgttaccac acacaagttt tgaggtgcatgcatggatgc cctgtggaaa 15580 gtttaaaaat attttggaaa tgatttgcat ggaagccatgtgtaaaacca tgacatccac 15640 ttggaggatg caataatgaa gaaaactaca aatttacatgcaactagtta tgcatgtagt 15700 ctatataatg aggattttgc aatactttca ttcatacacactcactaagt tttacacgat 15760 tataatttct tcatagccag cagatctaaa atg gct ccggat gcg gat aag ctt 15814 Met Ala Pro Asp Ala Asp Lys Leu 820 cga caacgc cag acg act gcg gta gcg aag cac aat gct gct acc ata 15862 Arg GlnArg Gln Thr Thr Ala Val Ala Lys His Asn Ala Ala Thr Ile 825 830 835 tcgacg cag gaa cgc ctt tgc agt ctg tct tcg ctc aaa ggc gaa gaa 15910 SerThr Gln Glu Arg Leu Cys Ser Leu Ser Ser Leu Lys Gly Glu Glu 840 845 850855 gtc tgc atc gac gga atc atc tat gac ctc caa tca ttc gat cat ccc15958 Val Cys Ile Asp Gly Ile Ile Tyr Asp Leu Gln Ser Phe Asp His Pro860 865 870 ggg ggt gaa acg atc aaa atg ttt ggt ggc aac gat gtc act gtacag 16006 Gly Gly Glu Thr Ile Lys Met Phe Gly Gly Asn Asp Val Thr ValGln 875 880 885 tac aag atg att cac ccg tac cat acc gag aag cat ttg gaaaag atg 16054 Tyr Lys Met Ile His Pro Tyr His Thr Glu Lys His Leu GluLys Met 890 895 900 aag cgt gtc ggc aag gtg acg gat ttc gtc tgc gag tacaag ttc gat 16102 Lys Arg Val Gly Lys Val Thr Asp Phe Val Cys Glu TyrLys Phe Asp 905 910 915 acc gaa ttt gaa cgc gaa atc aaa cga gaa gtc ttcaag att gtg cga 16150 Thr Glu Phe Glu Arg Glu Ile Lys Arg Glu Val PheLys Ile Val Arg 920 925 930 935 cga ggc aag gat ttc ggt act ttg gga tggttc ttc cgt gcg ttt tgc 16198 Arg Gly Lys Asp Phe Gly Thr Leu Gly TrpPhe Phe Arg Ala Phe Cys 940 945 950 tac att gcc att ttc ttc tac ctg cagtac cat tgg gtc acc acg gga 16246 Tyr Ile Ala Ile Phe Phe Tyr Leu GlnTyr His Trp Val Thr Thr Gly 955 960 965 acc tct tgg ctg ctg gcc gtg gcctac gga atc tcc caa gcg atg att 16294 Thr Ser Trp Leu Leu Ala Val AlaTyr Gly Ile Ser Gln Ala Met Ile 970 975 980 ggc atg aat gtc cag cac gatgcc aac cac ggg gcc acc tcc aag cgt 16342 Gly Met Asn Val Gln His AspAla Asn His Gly Ala Thr Ser Lys Arg 985 990 995 ccc tgg gtc aac gac atgcta ggc ctc ggt gcg gat ttt att ggt ggt 16390 Pro Trp Val Asn Asp MetLeu Gly Leu Gly Ala Asp Phe Ile Gly Gly 1000 1005 1010 1015 tcc aag tggctc tgg cag gaa caa cac tgg acc cac cac gct tac acc 16438 Ser Lys TrpLeu Trp Gln Glu Gln His Trp Thr His His Ala Tyr Thr 1020 1025 1030 aatcac gcc gag atg gat ccc gat agc ttt ggt gcc gaa cca atg ctc 16486 AsnHis Ala Glu Met Asp Pro Asp Ser Phe Gly Ala Glu Pro Met Leu 1035 10401045 cta ttc aac gac tat ccc ttg gat cat ccc gct cgt acc tgg cta cat16534 Leu Phe Asn Asp Tyr Pro Leu Asp His Pro Ala Arg Thr Trp Leu His1050 1055 1060 cgc ttt caa gca ttc ttt tac atg ccc gtc ttg gct gga tactgg ttg 16582 Arg Phe Gln Ala Phe Phe Tyr Met Pro Val Leu Ala Gly TyrTrp Leu 1065 1070 1075 tcc gct gtc ttc aat cca caa att ctt gac ctc cagcaa cgc ggc gca 16630 Ser Ala Val Phe Asn Pro Gln Ile Leu Asp Leu GlnGln Arg Gly Ala 1080 1085 1090 1095 ctt tcc gtc ggt atc cgt ctc gac aacgct ttc att cac tcg cga cgc 16678 Leu Ser Val Gly Ile Arg Leu Asp AsnAla Phe Ile His Ser Arg Arg 1100 1105 1110 aag tat gcg gtt ttc tgg cgggct gtg tac att gcg gtg aac gtg att 16726 Lys Tyr Ala Val Phe Trp ArgAla Val Tyr Ile Ala Val Asn Val Ile 1115 1120 1125 gct ccg ttt tac acaaac tcc ggc ctc gaa tgg tcc tgg cgt gtc ttt 16774 Ala Pro Phe Tyr ThrAsn Ser Gly Leu Glu Trp Ser Trp Arg Val Phe 1130 1135 1140 gga aac atcatg ctc atg ggt gtg gcg gaa tcg ctc gcg ctg gcg gtc 16822 Gly Asn IleMet Leu Met Gly Val Ala Glu Ser Leu Ala Leu Ala Val 1145 1150 1155 ctgttt tcg ttg tcg cac aat ttc gaa tcc gcg gat cgc gat ccg acc 16870 LeuPhe Ser Leu Ser His Asn Phe Glu Ser Ala Asp Arg Asp Pro Thr 1160 11651170 1175 gcc cca ctg aaa aag acg gga gaa cca gtc gac tgg ttc aag acacag 16918 Ala Pro Leu Lys Lys Thr Gly Glu Pro Val Asp Trp Phe Lys ThrGln 1180 1185 1190 gtc gaa act tcc tgc act tac ggt gga ttc ctt tcc ggttgc ttc acg 16966 Val Glu Thr Ser Cys Thr Tyr Gly Gly Phe Leu Ser GlyCys Phe Thr 1195 1200 1205 gga ggt ctc aac ttt cag gtt gaa cac cac ttgttc cca cgc atg agc 17014 Gly Gly Leu Asn Phe Gln Val Glu His His LeuPhe Pro Arg Met Ser 1210 1215 1220 agc gct tgg tat ccc tac att gcc cccaag gtc cgc gaa att tgc gcc 17062 Ser Ala Trp Tyr Pro Tyr Ile Ala ProLys Val Arg Glu Ile Cys Ala 1225 1230 1235 aaa cac ggc gtc cac tac gcctac tac ccg tgg atc cac caa aac ttt 17110 Lys His Gly Val His Tyr AlaTyr Tyr Pro Trp Ile His Gln Asn Phe 1240 1245 1250 1255 ctc tcc acc gtccgc tac atg cac gcg gcc ggg acc ggt gcc aac tgg 17158 Leu Ser Thr ValArg Tyr Met His Ala Ala Gly Thr Gly Ala Asn Trp 1260 1265 1270 cgc cagatg gcc aga gaa aat ccc ttg acc gga cgg gcg taa 17200 Arg Gln Met AlaArg Glu Asn Pro Leu Thr Gly Arg Ala 1275 1280 agatctgccg gcatcgatcccgggccatgg cctgctttaa tgagatatgc gagacgccta 17260 tgatcgcatg atatttgctttcaattctgt tgtgcacgtt gtaaaaaacc tgagcatgtg 17320 tagctcagat ccttaccgccggtttcggtt cattctaatg aatatatcac ccgttactat 17380 cgtattttta tgaataatattctccgttca atttactgat tgtccgtcga cgagctcggc 17440 gcgcctctag aggatcgatgaattcagatc ggctgagtgg ctccttcaac gttgcggttc 17500 tgtcagttcc aaacgtaaaacggcttgtcc cgcgtcatcg gcgggggtca taacgtgact 17560 cccttaattc tccgctcatgatcagattgt cgtttcccgc cttcagttta aactatcagt 17620 gtttgacagg atatattggcgggtaaacct aagagaaaag agcgtttatt agaataatcg 17680 gatatttaaa agggcgtgaaaaggtttatc cttcgtccat ttgtatgtgc atgccaacca 17740 cagggttccc ca 17752 29290 PRT Unknown Plant expression vector with 3 promoter- terminatorexpression cassettes with Physcomitrella elongase + desaturase +Phaeodactylum desaturase inserted 29 Met Glu Val Val Glu Arg Phe Tyr GlyGlu Leu Asp Gly Lys Val Ser 1 5 10 15 Gln Gly Val Asn Ala Leu Leu GlySer Phe Gly Val Glu Leu Thr Asp 20 25 30 Thr Pro Thr Thr Lys Gly Leu ProLeu Val Asp Ser Pro Thr Pro Ile 35 40 45 Val Leu Gly Val Ser Val Tyr LeuThr Ile Val Ile Gly Gly Leu Leu 50 55 60 Trp Ile Lys Ala Arg Asp Leu LysPro Arg Ala Ser Glu Pro Phe Leu 65 70 75 80 Leu Gln Ala Leu Val Leu ValHis Asn Leu Phe Cys Phe Ala Leu Ser 85 90 95 Leu Tyr Met Cys Val Gly IleAla Tyr Gln Ala Ile Thr Trp Arg Tyr 100 105 110 Ser Leu Trp Gly Asn AlaTyr Asn Pro Lys His Lys Glu Met Ala Ile 115 120 125 Leu Val Tyr Leu PheTyr Met Ser Lys Tyr Val Glu Phe Met Asp Thr 130 135 140 Val Ile Met IleLeu Lys Arg Ser Thr Arg Gln Ile Ser Phe Leu His 145 150 155 160 Val TyrHis His Ser Ser Ile Ser Leu Ile Trp Trp Ala Ile Ala His 165 170 175 HisAla Pro Gly Gly Glu Ala Tyr Trp Ser Ala Ala Leu Asn Ser Gly 180 185 190Val His Val Leu Met Tyr Ala Tyr Tyr Phe Leu Ala Ala Cys Leu Arg 195 200205 Ser Ser Pro Lys Leu Lys Asn Lys Tyr Leu Phe Trp Gly Arg Tyr Leu 210215 220 Thr Gln Phe Gln Met Phe Gln Phe Met Leu Asn Leu Val Gln Ala Tyr225 230 235 240 Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro Gln Trp Leu IleLys Ile 245 250 255 Leu Phe Tyr Tyr Met Ile Ser Leu Leu Phe Leu Phe GlyAsn Phe Tyr 260 265 270 Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly Lys GlnLys Gly Ala Lys 275 280 285 Thr Glu 290 30 525 PRT Unknown Plantexpression vector with 3 promoter- terminator expression cassettes withPhyscomitrella elongase + desaturase + Phaeodactylum desaturase inserted30 Met Val Phe Ala Gly Gly Gly Leu Gln Gln Gly Ser Leu Glu Glu Asn 1 510 15 Ile Asp Val Glu His Ile Ala Ser Met Ser Leu Phe Ser Asp Phe Phe 2025 30 Ser Tyr Val Ser Ser Thr Val Gly Ser Trp Ser Val His Ser Ile Gln 3540 45 Pro Leu Lys Arg Leu Thr Ser Lys Lys Arg Val Ser Glu Ser Ala Ala 5055 60 Val Gln Cys Ile Ser Ala Glu Val Gln Arg Asn Ser Ser Thr Gln Gly 6570 75 80 Thr Ala Glu Ala Leu Ala Glu Ser Val Val Lys Pro Thr Arg Arg Arg85 90 95 Ser Ser Gln Trp Lys Lys Ser Thr His Pro Leu Ser Glu Val Ala Val100 105 110 His Asn Lys Pro Ser Asp Cys Trp Ile Val Val Lys Asn Lys ValTyr 115 120 125 Asp Val Ser Asn Phe Ala Asp Glu His Pro Gly Gly Ser ValIle Ser 130 135 140 Thr Tyr Phe Gly Arg Asp Gly Thr Asp Val Phe Ser SerPhe His Ala 145 150 155 160 Ala Ser Thr Trp Lys Ile Leu Gln Asp Phe TyrIle Gly Asp Val Glu 165 170 175 Arg Val Glu Pro Thr Pro Glu Leu Leu LysAsp Phe Arg Glu Met Arg 180 185 190 Ala Leu Phe Leu Arg Glu Gln Leu PheLys Ser Ser Lys Leu Tyr Tyr 195 200 205 Val Met Lys Leu Leu Thr Asn ValAla Ile Phe Ala Ala Ser Ile Ala 210 215 220 Ile Ile Cys Trp Ser Lys ThrIle Ser Ala Val Leu Ala Ser Ala Cys 225 230 235 240 Met Met Ala Leu CysPhe Gln Gln Cys Gly Trp Leu Ser His Asp Phe 245 250 255 Leu His Asn GlnVal Phe Glu Thr Arg Trp Leu Asn Glu Val Val Gly 260 265 270 Tyr Val IleGly Asn Ala Val Leu Gly Phe Ser Thr Gly Trp Trp Lys 275 280 285 Glu LysHis Asn Leu His His Ala Ala Pro Asn Glu Cys Asp Gln Thr 290 295 300 TyrGln Pro Ile Asp Glu Asp Ile Asp Thr Leu Pro Leu Ile Ala Trp 305 310 315320 Ser Lys Asp Ile Leu Ala Thr Val Glu Asn Lys Thr Phe Leu Arg Ile 325330 335 Leu Gln Tyr Gln His Leu Phe Phe Met Gly Leu Leu Phe Phe Ala Arg340 345 350 Gly Ser Trp Leu Phe Trp Ser Trp Arg Tyr Thr Ser Thr Ala ValLeu 355 360 365 Ser Pro Val Asp Arg Leu Leu Glu Lys Gly Thr Val Leu PheHis Tyr 370 375 380 Phe Trp Phe Val Gly Thr Ala Cys Tyr Leu Leu Pro GlyTrp Lys Pro 385 390 395 400 Leu Val Trp Met Ala Val Thr Glu Leu Met SerGly Met Leu Leu Gly 405 410 415 Phe Val Phe Val Leu Ser His Asn Gly MetGlu Val Tyr Asn Ser Ser 420 425 430 Lys Glu Phe Val Ser Ala Gln Ile ValSer Thr Arg Asp Ile Lys Gly 435 440 445 Asn Ile Phe Asn Asp Trp Phe ThrGly Gly Leu Asn Arg Gln Ile Glu 450 455 460 His His Leu Phe Pro Thr MetPro Arg His Asn Leu Asn Lys Ile Ala 465 470 475 480 Pro Arg Val Glu ValPhe Cys Lys Lys His Gly Leu Val Tyr Glu Asp 485 490 495 Val Ser Ile AlaThr Gly Thr Cys Lys Val Leu Lys Ala Leu Lys Glu 500 505 510 Val Ala GluAla Ala Ala Glu Gln His Ala Thr Thr Ser 515 520 525 31 469 PRT UnknownPlant expression vector with 3 promoter-terminator expression cassetteswith + Phaeodactylum desaturase inserted 31 Met Ala Pro Asp Ala Asp LysLeu Arg Gln Arg Gln Thr Thr Ala Val 1 5 10 15 Ala Lys His Asn Ala AlaThr Ile Ser Thr Gln Glu Arg Leu Cys Ser 20 25 30 Leu Ser Ser Leu Lys GlyGlu Glu Val Cys Ile Asp Gly Ile Ile Tyr 35 40 45 Asp Leu Gln Ser Phe AspHis Pro Gly Gly Glu Thr Ile Lys Met Phe 50 55 60 Gly Gly Asn Asp Val ThrVal Gln Tyr Lys Met Ile His Pro Tyr His 65 70 75 80 Thr Glu Lys His LeuGlu Lys Met Lys Arg Val Gly Lys Val Thr Asp 85 90 95 Phe Val Cys Glu TyrLys Phe Asp Thr Glu Phe Glu Arg Glu Ile Lys 100 105 110 Arg Glu Val PheLys Ile Val Arg Arg Gly Lys Asp Phe Gly Thr Leu 115 120 125 Gly Trp PhePhe Arg Ala Phe Cys Tyr Ile Ala Ile Phe Phe Tyr Leu 130 135 140 Gln TyrHis Trp Val Thr Thr Gly Thr Ser Trp Leu Leu Ala Val Ala 145 150 155 160Tyr Gly Ile Ser Gln Ala Met Ile Gly Met Asn Val Gln His Asp Ala 165 170175 Asn His Gly Ala Thr Ser Lys Arg Pro Trp Val Asn Asp Met Leu Gly 180185 190 Leu Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Gln Glu Gln195 200 205 His Trp Thr His His Ala Tyr Thr Asn His Ala Glu Met Asp ProAsp 210 215 220 Ser Phe Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr ProLeu Asp 225 230 235 240 His Pro Ala Arg Thr Trp Leu His Arg Phe Gln AlaPhe Phe Tyr Met 245 250 255 Pro Val Leu Ala Gly Tyr Trp Leu Ser Ala ValPhe Asn Pro Gln Ile 260 265 270 Leu Asp Leu Gln Gln Arg Gly Ala Leu SerVal Gly Ile Arg Leu Asp 275 280 285 Asn Ala Phe Ile His Ser Arg Arg LysTyr Ala Val Phe Trp Arg Ala 290 295 300 Val Tyr Ile Ala Val Asn Val IleAla Pro Phe Tyr Thr Asn Ser Gly 305 310 315 320 Leu Glu Trp Ser Trp ArgVal Phe Gly Asn Ile Met Leu Met Gly Val 325 330 335 Ala Glu Ser Leu AlaLeu Ala Val Leu Phe Ser Leu Ser His Asn Phe 340 345 350 Glu Ser Ala AspArg Asp Pro Thr Ala Pro Leu Lys Lys Thr Gly Glu 355 360 365 Pro Val AspTrp Phe Lys Thr Gln Val Glu Thr Ser Cys Thr Tyr Gly 370 375 380 Gly PheLeu Ser Gly Cys Phe Thr Gly Gly Leu Asn Phe Gln Val Glu 385 390 395 400His His Leu Phe Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala 405 410415 Pro Lys Val Arg Glu Ile Cys Ala Lys His Gly Val His Tyr Ala Tyr 420425 430 Tyr Pro Trp Ile His Gln Asn Phe Leu Ser Thr Val Arg Tyr Met His435 440 445 Ala Ala Gly Thr Gly Ala Asn Trp Arg Gln Met Ala Arg Glu AsnPro 450 455 460 Leu Thr Gly Arg Ala 465

We claim:
 1. A method for the production of fatty acid esters with anincreased content of polyunsaturated fatty acids with at least twodouble bonds, which comprises introducing, into afatty-acid-ester-producing organism, at least one nucleic acid sequenceselected from the group consisting of a) a nucleic acid sequence withthe sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ IDNO: 11, b) nucleic acid sequences which, owing to the degeneracy of thegenetic code, are obtained by backtranslating the amino acid sequencesshown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12, c)derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5 or SEQ ID NO: 11, which encode polypeptides with theamino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6or SEQ ID NO: 12 and have at least 50% homology at the amino acid level,without essentially reducing the enzymatic action of the polypeptides,growing the organism, and isolating the fatty acid esters present in theorganism.
 2. A method as claimed in claim 1, wherein the fatty acidesters produced by the method comprise polyunsaturated C₁₈-, C₂₀- orC₂₂-fatty acid molecules with at least two double bonds in the fattyacid ester.
 3. A method as claimed in claim 1 or 2, wherein the C₁₈-,C₂₀- or C₂₂-fatty acid molecules are isolated from the organism in theform of an oil or lipid.
 4. A method as claimed in any of claims 1 to 3,wherein the organism is a microorganism, an animal or a plant.
 5. Amethod as claimed in any of claims 1 to 4, wherein the organism is atransgenic plant.
 6. A method as claimed in any of claims 1 to 5,wherein the fatty acid esters contain C₁₈-, C₂₀- or C₂₂-fatty acids withthree, four or five double bonds in the fatty acid ester.
 7. A method asclaimed in any of claims 1 to 6, wherein the polyunsaturated fatty acidscontained in the fatty acid esters are liberated.
 8. An isolated nucleicacid sequence encoding a polypeptide with desaturase activity, selectedfrom the group consisting of: a) a nucleic acid sequence with thesequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:11, b) nucleic acid sequences which, owing to the degeneracy of thegenetic code, are obtained by backtranslating the amino acid sequencesshown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12, c)derivatives of the nucleic acid sequences shown in SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5 or SEQ ID NO: 11, which encode polypeptides with theamino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6or SEQ ID NO: 12 and have at least 50% homology at the amino acid level,without essentially reducing the enzymatic action of the polypeptides.9. An amino acid sequence encoded by a nucleic acid sequence as claimedin claim
 8. 10. An amino acid sequence as claimed in claim 9 encoded bythe sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ IDNO:
 11. 11. A nucleic acid construct comprising a nucleic acid sequenceas claimed in claim 8, wherein the nucleic acid sequence is linked toone or more regulatory signals.
 12. A nucleic acid construct as claimedin claim 11, wherein additional biosynthesis genes of the fatty acid orlipid metabolism are present in the nucleic acid construct.
 13. Anucleic acid construct as claimed in claim 11 or 12, wherein thebiosynthesis gene of the fatty acid or lipid metabolism which is presentin the nucleic acid construct is a gene selected from the groupconsisting of acyl-CoA dehydrogenase(s), acyl-ACP[=acyl carrier protein]desaturase(s), acyl-ACP thioesterase(s), fatty acid acyl transferase(s),fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme Acarboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s),fatty acid acetylenases, lipoxygenases, triacylglycerol lipases,allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s).14. A vector comprising a nucleic acid sequence as claimed in claim 8 ora nucleic acid construct as claimed in claim
 11. 15. An organismcomprising at least one nucleic acid sequence as claimed in claim 8, atleast one nucleic acid construct as claimed in claim 11 or at least onevector as claimed in claim
 14. 16. An organism as claimed in claim 15,wherein the organism is a plant, a microorganism or an animal.
 17. Atransgenic plant comprising a functional or nonfunctional nucleic acidsequence as claimed in claim 8, a functional or nonfunctional nucleicacid construct as claimed in claim 11 or a functional or nonfunctionalvector as claimed in claim
 14. 18. The use of a nucleic acid sequence asclaimed in claim 8 or of a nucleic acid construct as claimed in claim 11for generating transgenic plants.
 19. An antibody which specificallybinds to a polypeptide encoded by one of the nucleic acids as claimed inclaim
 8. 20. An antisense nucleic acid molecule comprising thecomplementary sequence of the nucleic acid as claimed in claim
 8. 21. Anoil, lipid or fatty acid, or a fraction thereof, produced by the methodas claimed in any of claims 1 to
 7. 22. An oil, lipid or fatty acidcomposition, comprising an oil, lipid or fatty acid, or a fractionthereof, as claimed in claim
 21. 23. The use of an oil, lipid or fattyacid, or a fraction thereof, as claimed in claim 21 or of an oil, lipidor fatty acid composition as claimed in claim 22 in feed, foodstuffs,cosmetics or pharmaceuticals.
 24. A method of identifying an antagonistor agonist of desaturases, comprising a) contacting the cells whichexpress the polypeptide of the present invention with a candidatesubstance; b) testing the desaturate activity; c) comparing thedesaturase activity with a standard activity in the absence of thecandidate material, where an increase in the desaturase activity beyondthe standard indicates that the candidate material is an agonist and areduction in the desaturase activity indicates that the candidatematerial is an antagonist.
 25. A kit encompassing the nucleic acid asclaimed in claim 8, the nucleic acid construct as claimed in any ofclaims 11 to 13, the antibody as claimed in claim 19, the antisensenucleic acid molecule as claimed in claim 20, an antagonist or agonistidentified as claimed in claim 24, the composition as claimed in claim27, the amino acid sequence as claimed in claim
 9. 26. A kit as claimedin claim 25 comprising an oil, lipid or fatty acid, or a fractionthereof, as claimed in claim 21, or an oil, lipid or fatty acidcomposition as claimed in claim
 22. 27. A composition comprising theantibody as claimed in claim 19, the antisense nucleic acid construct asclaimed in claim 20 or an antagonist or agonist identified as claimed inclaim 24.